INTRODUCTION:
Chemically Phenyramidol hydrochloride is a-[(2-Pyridinylamino) methyl] benzene-methanol or 2-(b-hydroxyphenethylamino) pyridine. It is also be spelled as” fenyramidol”. It is available in the Indian market as tablet dosage form in the brand names of Cabral (Solvay), EzyFlex-400 etc. it is used therapeutically as an analgesic (Non-Narcotic) a as skeletal muscle relaxant. Highly Soluble in water, aqueous solutions are slightly acid and stable in ampoules. It is has a melting point of 140-142 0C. The mechanism of muscle relaxant effect of Phenyramidol hydrochloride is due to blocking of the inter-neuronal and polysynaptic reflexes. In narcotized and decelerated dogs, reflexes were effectively suppressed by Phenyramidol up to 100%.
Monosynaptic reflexes like the patellar and other idiopathic reflexes are not affected by Phenyramidol. Literature survey reveals that there is a no spectroscopic method available to estimate Phenyramidol Hydrochloride either for bulk drug or for tablet dosage forms. The present investigation was proposed to estimate Phenyramidol hydrochloride in bulk and tablet dosage form by Spectroscopic method which is rather simple, sensitive and may prove to be cost-effective. The method so developed was validated according to ICH guide lines for accuracy, precision and reproducibility, repeatability, robustness and ruggedness.
MATERIALS AND METHODS:
Material:
Phenyramidol Hydrochloride was obtained as a gift sample from M/S Kaytross ACG life Sciences limited, B-22, MIDC, Ambed, Nasik, India. Methanol was obtained S.D fine chemicals Pvt Ltd. Mumbai
Method:
Determination of absorption maxima:
Determination concentration range (Beer’s limit):
Phenyramidol hydrochloride stock solution (100µg/ml) was prepared using pure drug in distilleded water. Further dilutions were made using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml, 1.8ml, 2.0ml, 2.2ml and 2.4ml, of above solution was transferred to a series of 10ml volumetric flasks this gave a series of concentrations ranging from 2 to 24µg/ml of Phenyramidol hydrochloride.
The final volume was made up to 10ml mark using distilleded water, sonicated for 5 minutes. The resultant solutions were measured using a double beam UV-vis-spectrophotometer at a wave length of 236nm against a reagent blank. A calibration curve was plotted with concentration against absorbance. Results are shown in Fig. No 1 and Table No 1.
Preparation of standard calibration curve:
100 mg of pure Phenyramidol Hydrochloride drug was dissolved in a little quantity of distilled water in a 100 ml volumetric flask, the volume was made up to the mark using distilleded water. The solution was sonicated for 10 minutes. This gave a Phenyramidol hydrochloride solution with a concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted 100ml in volumetric flask using distilled water to obtain a concentration of 100µg/ml.
Further dilutions were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8ml solution was transferred to a series of 10ml volumetric flasks (to Obtain a series of concentrations ranging from 2-18 µg/ml of Phenyramidol hydrochloride). The final volume was made up to 10ml mark using distilleded water, sonicated for 5 minutes, the solutions were measured at a wave length of 236nm against a reagent blank. A calibration curve was plotted with concentration against absorbance. Results are shown in Fig. No 2,3 and Table No 2.
Estimation of Phenyramidol hydrochloride in tablet dosage forms:
Twenty tablets were weighed accurately and powdered. The Tablet powder equivalent to 50 mg of Phenyramidol hydrochloride was transferred into a 500mL volumetric flask and dissolved in a little quantity of distilleded water. Then the solution was sonicated for 30 minutes and filtered using Whatman filter paper No#41. The filtrate so obtained was diluted with distilleded water to produce 500ml. Further dilutions were made with distilleded water to get required concentrations within Beer’s - Lambert limits. The resultant solutions were measured at a wave length of 236nm against a reagent blank. The concentration of drug was calculated with the help of standard calibration curve. Results are shown in Table No 3.
ANALYTICAL METHOD VALIDATION 1-12
To validate analytical procedures:
Linearity and Range:
The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. The linearity was found to be 2-18µg/ml for the proposed colorimetric method. Optimum conditions, Optical characteristics and Statistical data of the Regression equation in UV-Vis method. Results are shown in Table No 10.
Accuracy and Precision:
The Accuracy of analysis was determined by performing recovery studies by spiking different concentrations of pure drug in the pre-analyzed tablet samples within the analytical concentration range of the proposed method at three different sets at the level of 80%, 100%, and 120%.
Here tablet dosage form containing Phenyramidol hydrochloride was used (EzyFlex-400). The procedure involved preparation of a stock solution of the tablet dosage form using suitable solvent and after appropriate dilution (within Beer’s limit) and absorbencies were measured at 236nm against reagent blank. To the above solutions, a pure drug of Phenyramidol hydrochloride (solution) was added to three sets at a level of 80%, 100% and 120%. After proper mixing, absorbencies were measured at 236nm and the results were calculated in terms of %RSD. Results are shown in Table No 5.
Precision:
Intra-day Precision:
Variation of results within the same day was analyzed. Intra-day precision was determined by analyzing the standard solutions of Phenyramidol hydrochloride (8, 10 and 12µg/ml) at three different time intervals of the same day i.e. morning, afternoon and evening. The concentration range selected on the basis of linearity range.
a) Inter-day Precision:
Variation of results between the days was analyzed. Inter-day precision was determined by analyzing the Phenyramidol hydrochloride (8, 10 and 12µg/ml) in linearity range at three consecutive days. Both inter and intra-day precisions were carried out thrice and average of three determinations was taken as mean S.D and calculated in terms of % Correlation coefficient
Reproducibility:
Standard solutions of Phenyramidol hydrochloride 4 to 36µg/ml were prepared and analyzed calorimetrically by Analyst 1 and Analyst 2, separately. The values so obtained were evaluated using F-test and t-test to verify their reproducibility.
Repeatability:
Standard solutions of Phenyramidol hydrochloride 2-18µg/ml were prepared and analyzed calorimetrically. The solutions were analyzed six times and the mean standard deviation (S.D) was calculated.
Ruggedness:
For determination of ruggedness, the degree of reproducibility of results was studied through changing the laboratory, instrument, glass apparatus and reagents. For this, Dr.H.L. T College of Pharmacy, Kengal, Channapatna was approached. It was found that the instrument, chemicals and reagents were of different brands and these differences were utilized to study the ruggedness of the proposed method. There were no significant variations in the analytical data when compared to establish method. Two different brands of tablets were analyzed used and the results were calculated in terms of % Recovery studies and expressed as %RSD. Results are shown in Table No and 8.
Robustness:
Robustness of the method was validated by changing the concentration of reagent, PH and temperature of reaction mixture before their estimation.
· The temperature of the reaction mixture was either raised by +50C (by warming) or reduced by -50C (by cooling) to study the effect of change in temperature over colour produced or on its absorption.
· To study the effect of change in PH, the reaction mixture was treated with 2 drops of 0.1N NaOH and the other with 0.1N HCl solution.
The experiments were repeated thrice and the data was used to calculate mean and standard deviation
Limit of detection and quantitation:
Limits of detection (LOD):
The LOD of the pure drug was estimated by analyzing the sample using a series of (very dilute solutions) and under same experimental conditions. First the dilute solutions (several times less than the lowest concentration of Beer range) were prepared and scanned over the optimum wave length of 236nm. The lowest concentration of the analyte which showed absorption at given λmax was recorded and the experiment was repeated thrice for confirmation. The LOD for pure drug was found to be 1.224µg/ml. Results are shown in Table No 9.
Limits of quantitation (LOQ):
The first approach is to evaluate it by visual evaluation and may be used for non instrumental methods and instrumental methods. LOQ is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantitated with acceptable accuracy and precision.
The second approach determines the signal - to - noise ratio by comparing measured signals from samples with known low concentrations of analyte with those of blank samples. LOQ is the minimum concentration at which the analyte can be reliably quantified at the signal - to - noise ratio of 10:1.
The third approach estimates is by using the equation;
LOQ=
The slope S may be estimated from the calibration curve of the analyte. The value of ş may is estimated by calculating the standard deviation of the responses obtained from the measurement of the analytical background response of an appropriate number of blank samples or calculating the residual standard deviation of the regression line from the calibration curve using samples containing the analyte in the range of the LOQ.
Whatever approach is applied, the LOQ should be subsequently validated by the analysis of a suitable number of samples prepared at the LOQ and determining the precision and accuracy at this level. Experimental determination of LOQ was found to be 5.222µg/ml
RESULTS:
Determination of beer’s limit:
Phenyramidol Hydrochloride stock solution (100µg/ml) was prepared using pure drug in distilleded water. Further dilutions were made using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml, 1.8ml, 2.0ml, 2.2ml and 2.4ml of above solution was transferred to a series of 10ml volumetric flasks and the volumes were made up to 10ml mark using distilled water and the content of the flask was shaken for 5 minutes, sonicated for 5 minutes, this gave a series of concentrations ranging from 2 to 24µg/ml of Phenyramidol hydrochloride.
The resultant solutions were measured at wave length of 236nm against a reagent blank. A calibration curve was plotted with concentration against absorbance From the graph it was clear that Beer’s law was obeyed in concentration range of 2-18µg/ml and deviation was observed above these concentrations. Results are shown in Fig. No 1 and Table no 1.
Fig. No.1 Beer’s limit for Phenyramidol Hydrochloride at 236nm
Table: 1 Beer’s range for Phenyramidol Hydrochloride:
|
Sl. No. |
Concentration in µg/ml |
Absorbance ± S. D* at λmax 236nm |
|
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. |
0.0 2 4 6 8 10 12 14 16 18 20 22 24 |
0.000 0.108±0.125 0.205±0.214 0.310±0.157 0.400±0.254 0.510±0.214 0.605±0.236 0.694±0.142 0.792±0.289 0.870±0.214 0.918±0.258 1.012±0.147 1.075±0.369 |
S.D*=standard deviation (n=3)
Determination of absorption maxima:
100 mg of pure Phenyramidol hydrochloride drug was dissolved in little quantity of distilleded water in a 100 ml volumetric flask, the volume was made up to the mark using distilleded water. The solution was sonicated for 10 minutes. This gave a Phenyramidol hydrochloride solution with concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted 100ml in volumetric flask using distilleded water to obtain a concentration of 100µg/ml.
Further dilutions were made using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml and 1.8ml of above solution was transferred to a series of 10ml volumetric flasks (to obtain a series of the concentrations ranging from 2-18µg/ml of Phenyramidol hydrochloride). The volumes were made up to 10 ml mark using distilleded water and the content of the flask were sonicated for 5 minutes. The resultant solutions were measured at wave length of 236nm against a reagent blank. A calibration curve was plotted with concentration against absorbance Results are shown in Fig. No 2 and Table No 2
Fig 2 Standard calibration curve for Phenyramidol Hydrochloride
Table 2: Absorbance of Phenyramidol Hydrochloride at various concentrations:
|
Sl. No. |
Concentration in µg/ml |
Absorbance±S.D*at λmax 236nm |
|
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. |
0.0 2 4 6 8 10 12 14 16 18 |
0.000 0.108±0.125 0.205±0.214 0.310±0.157 0.400±0.254 0.510±0.214 0.605±0.236 0.694±0.142 0.792±0.289 0.870±0.214
|
S.D*=standard deviation (n=3)
Standard calibration curve of phenyramidol hydrochloride:
100 mg of pure Phenyramidol hydrochloride drug was dissolved in little quantity of distilleded water in a 100 ml volumetric flask, the volume was made up to the mark using distilleded water. The solution was sonicated for 10 minutes. This gave a Phenyramidol hydrochloride solution with concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted 100ml in volumetric flask using distilleded water to obtain a concentration of 100µg/ml. Further dilutions were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8ml solution was transferred to a series of 10ml volumetric flasks (to Obtain a series of concentrations ranging from 2-18µg/ml of Phenyramidol hydrochloride).
The final volume was made up to 10ml mark using distilleded water, sonicated for 5 minutes, the solutions were measured at wave length of 236nm against a reagent blank. A calibration curve was plotted with concentration against absorbance. Results are shown in Fig. No 2 and 3 and Table No 2.
Figure 3. Absorption maxima of Phenyramidol hydrochloride at 236nm:
Estimation of phenyramidol hydrochloride in tablet dosage form:
Twenty tablets containing phenyramidol Hydrochloride were weighed accurately and powdered. The Tablet powder equivalent to 50mg of Phenyramidol hydrochloride was transferred into a 500mL volumetric flask and dissolved in little quantity of distilleded water. Then the solution was sonicated for 30 minutes and filtered using whatman filter paper No#41. The filtrate so obtained was further diluted with distilleded water to get a required concentration within Beer’s - Lambert limits (from above stock solution 1ml was diluted to 100ml which gives a concentration of 100µg/ml), From the above stock solution of drug, different aliquots were withdrawn and after appropriate dilution, sonicated for 5 minutes. The resultant solutions were measured at wave length of 236nm against a reagent blank. The concentration of drug was calculated with the help of standard calibration curve Results are shown in Table No 3.
Table 3: Absorption of drug from tablet dosage form:
|
Volume of stock solution used |
Amount of drug (label claim) (µg/ml) |
Absorbance at 236nm |
Amount of drug found (µg/ml) |
Percentage purity found±S.D* (%w/w) |
|
0.2ml 0.6ml 1.0ml 1.4ml 1.8ml |
2 6 10 14 18 |
0.1088 0.312 0.509 0.693 0.877 |
2.014 6.038 9.980 13.979 18.144 |
100.70±0.11 100.63±1.41 99.80±0.61 99.85±0.46 100.80±1.88 |
S.D*=Standard deviation (n=3)
Determination of accuracy:
Accuracy of analysis was determined by performing recovery studies by spiking different concentrations of pure drug in the pre-analyzed samples contained the drug within the analytical concentration range of the proposed method at three different set at level of 80%, 100% and 120%. Here marketed tablets (EzyFlex-400) were used; the procedure involved preparation of stock solution of the tablet dosage form using suitable solvent (distilled water) and after appropriate dilution (within Beer’s limit), absorbencies were measured at 236nm against regent blank. To the above solutions, pure drug of Phenyramidol hydrochloride (solution) was added at three sets at level of 80%, 100% and 120%. After proper mixing, absorbencies were re-measured at 236nm and the results were calculated in terms of %RSD. This experiment was repeated for six times and mean and standard deviations were considered. The result shows that there was no interference of common excipients present in the tablets. Results are shown in Table No 4.
Table 4: Accuracy results for Phenyramidol Hydrochloride:
|
Brands |
Initial amount (µg/ml) |
Amount of pure drug added (µg/ml) |
Amount recovered (µg/ml) |
% Recovery ± S. D* |
|
EzyFlex-400 |
5 5 5 |
8(80%) 10(100%) 12(120%) |
8.024 9.894 12.023 |
100.30±0.142 98.94±0.224 100.19±0.414 |
S.D* = Standard deviation (n=6)
Determination of precision:
It is the reproducibility of a result observed when the same quantity of one sample is repeatedly introduced in the instruments.
Intra-day assay is evaluated by analyzing aliquots of a homogeneous material several times on the same day with the same instrument. Inter-day assay is evaluated by analyzing aliquots of homogeneous material several times on different days. Variation of results within the same day was analyzed. Intra-day precision was determined by analyzing the standard solutions of Phenyramidol Hydrochloride (8, 10 and 12µg/ml) at three different time intervals of the same day i.e. morning, afternoon and evening. The concentration range selected on the basis of linearity range.
Variation of results between the days was analyzed. Inter-day precision was determined by analyzing the Phenyramidol Hydrochloride (8, 10 and 12µg/ml) in linearity range at three consecutive days.
Both inter and intra-day precisions were carried out thrice and average of three determinations was taken as mean S.D and calculated in terms of % Correlation coefficient. The % C. V values are less than 1 which indicated that there was no influence of time over the absorbencies of the drug. Results are shown in Table No 5
Table 5: Precision results for Phenyramidol HCl:
|
Sl. |
Conc. In (µg/ml) |
Inter-day absorbance Mean±S.D* |
% C.V |
Intra-day absorbance Mean±S.D* |
% C.V |
|
1. 2. 3. |
8 10 12 |
0.410±0.002 0.509±0.023 0.606±0.016 |
0.35 0.34 0.60 |
0.409±0.014 0.509±0.018 0.605±0.024 |
0.62 0.23 0.24 |
S.D*=Standard deviation, n=3
Determination of ruggedness parameters:
For determination of ruggedness, the degree of reproducibility of results was studied through changing the laboratory, instrument, glass apparatus and reagents. For this, Dr.H.L. T College of Pharmacy, Kengal, Channapatna was approached. It was found that the instrument, chemicals and reagents were of different brands and these differences were utilized to study the ruggedness of the proposed method. Tablets (brand name: EzyFlex-400) were powdered and after extraction and suitable dilution, the solutions were analyzed at same nanometer and the experiments were repeated for six times and the results were calculated in terms of % Recovery studies and expressed as %RSD. It was found that there was no significant variation in the analytical data when compared to establish method. Results are shown in Table No 6 and 7.
Determination of robustness:
Robustness of the method was validated by changing the concentration of reagent, PH and temperature of reaction mixture before their estimation.
The temperature of the reaction mixture was either raised by +50C (by warming) or reduced by -50C (by cooling) to study the effect of change in temperature over colour produced or on its absorption.
To study the effect of change in PH, the reaction mixture was treated with 2 drops of 0.1N NaOH and the other with 0.1N HCl solution. The experiments were repeated thrice and the data did not show any significant change in absorption pattern of the analyte. Thus it was established that a slight deliberate variations such as; ionic concentration of the reactants, PH and temperature does not having any major influence over the absorption pattern. Results are shown in Table No 8. Results of calibration data are shown in Table No 9.