INTRODUCTION:
Mebendazole (MEB) chemically is {(5-Benzoyl-1H-benzimidazole-2-yl)-carbamic acid-methyl ester}1,2. Mebendazole irreversibly blocks the uptake of exogenous glucose by nematodes, leading to glycogen depletion and reduce generation of ATP required for survival. Due to this the parasites dies or are slowly immobilized and clear from the gut gradually. Mebendazole is drug of choice in enterobiasis and trichuriasis.
Pyrental pamoate (PYR) chemically is {E1, 4, 5, 6 tetrahydro-1-methyl-2[-2-(2-triethyl)ethenyl]pyrimidine}1,2. Pyrantal pamoate is highly effective for the treatment of oxyuriasis, ascariasisi and trichostronglylus oriental is infectious. The anthelmentic action of pyrental pamoate is due to the inhibition of neuromuscular transmission. It causes a spastic neuromuscular paralysis in the worm, which is subsequently expelled from the host infection.
Literature survey revealed that various method has been reported for estimation of MEB and PYR individually viz. HPLC3,4,5 GC6, HPTLC7, Colorimetric8, UV-Spectrophotometric9, TLC10,11,12.
The method reported for simultaneous estimation of both these drug are by vierodt’s method13, RP-HPLC14,15, HPTLC16. In present project attempt was made to develop alternative simple, rapid, accurate and reproducible, UV-spectrophotometric method for simultaneous estimation of MEB and PYR in fixed dose combination tablet base on different quantities spectrophotometric approaches.
Methods and Materials
Chemicals and Instruments:
All the chemicals used in the development of methods are of analytical grades. UV-Spectrophotometer used in the study is Shimadzu UV-2401 double beam spectrophotometer with 1.0 cm matched pair of rectangular quartz cells were used for spectrophotometric studies.
Standard Solution:
Stock standard solution:
An accurately weighed quantity of MEB (≈ 50 mg) and PYR (≈ 50 mg) was dissolved in 20.0 mL of DMF and volume was made up to 50.0 mL with methanol (1000µg/mL).
Selection of wavelengths:
The aliquots portions of stock standard solution of MEB and PYR were diluted appropriately with methanol to obtain a concentration 10µg/mL of each drug. The solutions were scanned in the range of 400-200 nm in 1.0 cm cell against blank.
Study of Beer-Lambert’s Law:
The aliquot portions of stock standard solutions of MEB and PYR were diluted with methanol to get the series of concentration from 0-3µg/mL of both the drugs. Similarly aliquot portions of stock standard solutions were mixed and diluted appropriately to get series of concentration ranging from 0-30 µg/mL. The absorbances of each solution were measured at the selected wavelengths.
Additivity Study:
Six standard solution of different ratio were prepared and absorbances of each of six solutions were measure at the selected wavelengths against blank. The observed absorbance of the prepared solution at respective wavelength were added and the resultant theoretical total absorbance were compared with practically observed absorbance of solution i.e. absorbance of mixture. The aliquot portion of MEB and PYR stock were diluted with methanol to obtain accurately about 10.0 µg/mL of each MEB and PYR. The absorbances of resulting solutions were measured at the selected wavelength against blank. The E(1%, 1cm) values were calculated using formula given below.
E(1%, 1cm) = Absorbance
Concentration (g/100 mL)
Analysis of Laboratory Mixture:
In order to establish suitability of the proposed method for simultaneous estimation of MEB and PYR in pharmaceutical formulation, the method was first tried for the estimation of the components in a standard laboratory mixture of two drugs.
Accurately weighed quantities of MEB and PYR (≈ 75 mg and ≈50 mg respectively) were mixed and dissolved by vigorous shaking in 20.0 mL of DMF in 50.0 mL volumetric flask and volume was made upto the mark by methanol. An accurately measured 5.0 mL portion of the resultant solution was diluted to 50.0 mL with methanol. A 5.0mL portion was further diluted to 50.0 mL with methanol. The absorbance of the final solution was measured at the selected wavelengths in 1.0 cm cell against blank.
Amount of each drug was calculated using following formulae
Cx =
Qm-Qy A
Qx-Qy ax1
Cy =
Qm-Qx A
Qy-Qx ay1
Where, Qm = Ratio of absorbance of mixture at 311.6 nm and 281.0 nm
Qy = Ratio of absorptivity of PYR at 311.6 nm and 281.0 nm = 0.3808
Qx = Ratio of absorptivity of MEB at 311.6 nm and 281.0 nm = 1.6656
A = Absorbance of mixture of 281.0 nm
ax1 and ay1= Absorptivity of MEB and PYR at 281.0 nm respectively = 309.30
Cx and Cy = Concentration in g/100 mL of MEB and PYR respectively in final solution.
The drug can be estimated by the formula
% Estimation of
drug = ×100 1100100
C×V×100
 |
W
Where, C = Cx and Cy
V = Volume of stock = 50.0 mL
W = Weight of respective drug either MEB or PYR in laboratory mixture
Analysis of Marketed Formulation:
Twenty tablets were weighed and finely powdered. An accurately weighed quantity of tablets powder equivalent to about 75 mg of MEB (≈ 50 mg of PYR base) was taken in 50.0 mL volumetric flask and dissolved in 20.0 mL of DMF with vigorous shaking and volume was made to 50.0 mL with methanol. The solution was then filtered through whatman filter paper. The aliquot portion of the filtrate was further diluted to get final concentration of about 15 µg/mL of MEB (and also10 µg/mL of PYR) on the basis of labeled claim. The absorbance of sample solution was read at 281.0 nm and 311.6 nm. The concentration of drugs in final solution (Cx and Cy) were calculated by using same formula as in the laboratory mixture and percent labeled claims were calculated by using formula
Where,
Cx and Cy = Concentration in g/100 mL of MEB and PYR in final solution respectively
D = Dilution factor = 100
Wm = Weight of tablet powder taken in g.
L = Labeled claim of drug in g/tablet
(MEB = 0.150 g/tab, PYR base = 0.100 g/tab)
Mb = Molecular weight of Pyrantal base = 206.31
Mp = Molecular weight of Pyrantal pamoate = 594.68
Recovery Study:
Mixed standard solutions were prepared by dissolving accurately 150 mg of PYR and 75 mg of MEB in about 50 mL of DMF by vigorous shaking and volume was made upto 1000.0 mL with DMF. A 10.0 mL aliquot of the mixed standard solution (containing accurately about 7.5 mg MEB and 15.0 mg PYR) was transferred to 50.0 mL flask containing accurately weighed, preanalysed tablet powder equivalent to about 70 mg of MEB followed by addition of 10.0 mL of DMF. The mixture was vigorously shaken and volume was made upto mark with methanol. The solution was filtered and the aliquot of the filtrate was further 100 times diluted and the absorbances were read at 281.0 nm and 311.6 nm. The total amounts of drugs (Cx and Cy) were calculated by the same formula given under estimation of tablet.
The percent recovery was then calculated by using formula
Where, T = Total drug estimated
C = Amount of drug contributed by tablet powder on preanalysed basis
P = Amount of pure drug added
Validation of method:
Accuracy:
Accuracy of each of the proposed method was ascertained on the bsis of recovery studies performed by standard addition method.
Precision:
Precision of the analytical method is expressed as the series of the measurement. It was ascertained by replicate estimation of the drugs by the proposed methods.
Specificity:
Accurately weighed quantities of the tablets powder equivalent to about 38 mg of MEB were taken in a dry 50.0 mL volumetric flask. Each sample was then store for 24 hrs under the following different conditions.
1. At room temperature (normal)
2. At 50° C after addition of 1 mL of 0.1 M NaOH (Alkali)
3. At 50° C after addition of 1 ml of 0.1 M HCl (acid)
4. At 50° C after addition of 1 mL of 3% H2O2 (oxide)
5. At 60° C (heat)
After 24 hr each treated sample was dissolved in 20.0 mL DMF with vigorous shaking and volume was made upto the mark with methanol. The solutions were then filtered and aliquots of filtrate were 100 times diluted with methanol. Absorbances were read at 281 nm and 311.6 nm. The percent labeled claims were calculated by the method using formula under estimation of MEB and PYR by proposed method.
Linearity and Range:
Accurately weighed quantities of tablet powder equivalent to 60, 67.5, 75 mg (representing 80, 90 and 100% label claim of MEB were taken in three different 50.0 mL volumetric flasks and were dissolved in 20.0 mL of DMF with vigorous shaking. The volume were made upto the mark with methanol.
A 10.0 mL and 20.0 mL aliquots of mixed standard solution were transferred to 50.0 mL volumetric flaks, each containing tablet powder equivalent to about 75 mg of MEB (representing 110, 120% label claim) followed by addition of 10.0 mL of DMF. After vigorous shaking volumes were made upto the mark with methanol. The solutions were then filtered and aliquots of filtrate were 100 times diluted with methanol. Absorbances were read at 281.0 nm and 311.6 nm.
Ruggedness:
Ruggedness was ascertained by getting the sample analysed from different analysts and carrying out analysis on different days and in different laboratory by proposed method.
Results and Discussion:
The attempt was made to develop an alternative and economical method for simultaneous estimation of Mebendazole and Pyrantal pamoate by isobestic point method.
Overlain spectra of MEB and PYR had revealed that both the drugs have same absorbance (isobestic point) in 40% dimethy formamide (methanol) at 281.0 nm. Mebendazole shows its absorption maxima at 311.6 nm in same solvents. Hence it was predicted that simultaneous estimation of both drugs should be possible by solving a pair of equation for isobestic point method, by measuring the absorption of sample solution at 281.0 nm and 311.6 nm.
Both the drugs MEB and PYR and its mixture follow beer-lambert’s law in the range of 0-30 µg/mL at all the selected wavelengths (Figure 1). Mixture of drugs showed Additivity of absorbance at selected wavelengths indicating that there is no interaction between the drugs in 49% v/v DMF in methanol. Specific absorptivity values determined for both the drugs were 308.9±0.93 and 515±1.59 for MEB and 309.0±0.54 and 117.9±0.30 for PYR at 281.0 nm and 311.6 nm respectively (Table 1).
FIGURE 1
Table- 1 (a): Absorbtivity values for MEB
|
Sr. No. |
Conc. Of MEB (g/100 ml) |
Absorbance at |
E (1%, 1 cm) at |
||
|
281.0 nm |
311.6 nm |
281.0 nm |
311.6 nm |
||
|
1 |
0.001014 |
0.3129 |
0.5232 |
309.37 |
515.68 |
|
2 |
0.000997 |
0.3094 |
0.5141 |
310.09 |
515.33 |
|
3 |
0.001002 |
0.3089 |
0.5178 |
308.24 |
516.79 |
|
4 |
0.000994 |
0.3072 |
0.5102 |
309.06 |
512.58 |
|
5 |
0.000900 |
0.2771 |
0.4643 |
307.73 |
515.26 |
|
Mean ± SD |
308.89± 0.93 |
512.26±1.59 |
|||
Table- 1 (b): Absorbtivity values for PYR
|
Sr. No. |
Conc. Of PYR (g/100 ml) |
Absorbance at |
E (1%, 1 cm) at |
||
|
281.0 nm |
311.6 nm |
281.0 nm |
311.6 nm |
||
|
1 |
0.001033 |
0.3207 |
0.1220 |
310.38 |
118.14 |
|
2 |
0.000963 |
0.2981 |
0.1135 |
309.49 |
117.52 |
|
3 |
0.001052 |
0.3264 |
0.1240 |
310.02 |
117.99 |
|
4 |
0.001025 |
0.3177 |
0.1212 |
309.71 |
118.05 |
|
5 |
0.001006 |
0.3109 |
0.1183 |
308.95 |
117.37 |
|
Mean ± SD |
309.71± 0.54 |
117.87±0.30 |
|||
The percent estimation of mixed drugs in laboratory mixture was done to be 100.30±0.19 and 99.32±0.78 for MEB and PYR respectively (Table 2). The percent drug estimation in marketed formulation was found to be 100.30±0.34 and 101.45±1.84 (Table 3). The method was validated according to ICH guidelines. The accuracy of method was validated by percent recovery of both the drugs. The average percent recovery was found to be 100.45±0.23 and 100.98±0.60 (Table 4). The results of the method lies within the prescribed limit of 98-102 % show that method is free from interference from Excipients. The next parameter studied was precision of the experimental method. The replicate estimation of both MEB and PYR in same batch of tablet was analyzed by the proposed method and showed quite concurrent results indicating reliability of the method. The values ± SD or RSD and coefficients correlation are within the prescribed limit of 2% showing high precision of the method.
The other parameters studied were specificity, linearity and ruggedness. This study was carried out to check degradation and stability of the drug (Figure 2, Table 5). In the specificity study the samples were exposed to different stress conditions like acid, alkali, oxide, heat and uv-visible lights. The MEB shows slight degradation of drug under alkali and acidic condition. But PYR shows degradation in alkali and uv-visible light (Table 6). Overall this method is incapable of finding exact degradation of drugs. The linearity and range study were also carried out over the range of 80-120% of labeled claim for both the drug in formulation the percent lable claim verses absorbance plots shows a linear relationship with correlation coefficients very closer to 1. Ruggedness studies were carried out under three different conditions i.e., different day, analyst and laboratories shows that the results of estimation by proposed method are very much reproducible under variety of conditions (Table 7).
Table-2: Estimation of drug in Laboratory Mixture
|
Sr. No. |
Wt. of MEB |
Wt. of PYR |
Absorbance of Mixture |
% Drug Estimated |
||
|
281.0 nm |
311.6 nm |
MEB |
PYR |
|||
|
1 |
0.07527 |
0.04456 |
0.7578 |
0.8814 |
100.03 |
98.11 |
|
2 |
0.07572 |
0.04352 |
0.7356 |
0.8820 |
99.84 |
99.03 |
|
3 |
0.07516 |
0.04308 |
0.7361 |
0.8853 |
99.92 |
99.74 |
|
4 |
0.07720 |
0.04012 |
0.7303 |
0.8997 |
100.20 |
99.55 |
|
5 |
0.07566 |
0.04202 |
0.7320 |
0.8878 |
100.30 |
100.16 |
|
Mean |
100.06 |
99.32 |
||||
|
± S. D. |
0.19 |
0.78 |
||||
|
C. V. |
0.19 |
0.79 |
||||
|
R. S. D. |
0.001 |
0.007 |
||||
Table-3: Estimation of drugs in Tablet
|
Sr. No. |
Wt. Sample (g) |
Absorbance of Mixture |
% Drug Estimated |
||
|
281.0 nm |
311.6 nm |
MEB |
PYR |
||
|
1 |
0.2896 |
1.4011 |
1.1382 |
100.88 |
103.78 |
|
2 |
0.2866 |
1.3598 |
1.1109 |
99.98 |
100.18 |
|
3 |
0.2870 |
1.3512 |
1.1092 |
100.14 |
99.08 |
|
4 |
0.2869 |
1.3790 |
1.1202 |
100.23 |
102.13 |
|
5 |
0.2875 |
1.3802 |
1.224 |
100.29 |
101.45 |
|
Mean |
100.30 |
101.45 |
|||
|
± S. D. |
0.34 |
1.84 |
|||
|
C. V. |
0.34 |
1.84 |
|||
|
R. S. D. |
0.003 |
0.018 |
|||
With regards to obtained results, proposed method revealed that the experimental study signifies simple, accurate, fast, precise and reproducible isobestic point method for simultaneous estimation of MEB and PYR in their combined dosage form and can be used for routine analysis of both the drug in commercially available marketed formulation.
Acknowledgement:
The authors are thankful to Head, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for providing laboratory facilities. Authors are also thankful to M/S Cipla Ltd Mumbai and IPCA laboratories Ltd. Ratlam respectively.