Method Development and its Validation for Simultaneous estimation of Levamisole Hydrochloride and Mebendazole as API and in Combination in Tablet Dosage Form by Simultaneous Equation Method of UV Spectrophotometry

Vaibhav Jain*¹, Rajkumar Chauhan¹, B. P. Nagori¹, Sabir Anis²& Ranjan Kumar Singh³

1-Lachoo Memorial College of Science & Technology (Autonomous) Pharmacy Wing, Jodhpur - 342001, Rajasthan, India.

2-Mualana Azad University, Jodhpur, Rajasthan, India.

 3- School of pharmacy, Dept. of Pharmacology, Neemrana, Alwar, Rajasthan-341705.

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*Correspondence: vaibhavs.jain96@gmail.com

INTRODUCTION

Mebendazole (MBZ) is methyl (5-benzoyl-1Hbenzimidazol-2-yl) carbamate. It is soluble in formic acid, shown in Figure 1 (a). Levamisole Hydrochloride (LVM) is (S)-6-phenyl-2, 3, 5, 6-tetrahydroimidazo [2,1-b] thiazole hydrochloride Figure 1 (b). It is soluble in methanol, ethanol, and water. MBZ and LVM are official in Indian Pharmacopoeia, British Pharmacopoeia, European Pharmacopoeia, and United States Pharmacopoeia. Both drugs are used as anthelmintics [1].

Figure 1 (a) : Structure of Mebendazole

Figure 1(b) : Structure of Mebendazole

Literature review revealed that various analytical methods like first-order derivative spectroscopy, and Q absorbance spectroscopy have been reported for single drugs or in combination with other drugs [2-8]. However, the simultaneous equation method has not been reported for the simultaneous estimation of the combination of mebendazole and levamisole hydrochloride.[9-14].

 MATERIALS AND METHODS:

Shimadzu model 1800 double beam UV-visible spectrophotometer with a spectral width of 2 nm, wavelength accuracy of 0.5 nm, and a pair of 10 mm matched quartz cells was used to measure absorbance. The instrument was equipped with Shimadzu UV Probe 2.34 version software. The MBZ reference standard was gifted by Baroque Pharmaceuticals Pvt. Ltd. (Cambay, Gujarat - India) and the LVM reference standard was received as a gift sample from Shree Pharma (Gujarat, India). The commercial fixed-dose formulation containing 100 mg MBZ and 150 mg of LVM, RAID Tablet was procured from the local market. Analytical grade Methanol (Loba Chemicals) and H2SO4 (Loba Chemicals) were procured from the campus laboratory.

 Preparation of stock solutions and standard solutions for determination of λmax:

Accurately weighed 10mg each of MBZ and LVM were transferred to two different 100 mL volumetric flasks. The volume was made up to the mark with 1 % v/v H2SO4 in methanol to obtain stock solutions of MBZ and LVM having a concentration of 100 μg/mL of each. From the above stock solutions, aliquots of 1 mL were pipetted out and placed into two different 10 mL volumetric flasks. The volume was made up to mark with 1 % v/v H2SO4 in methanol to give solutions containing 10 μg/mL of MBZ and LVM. The absorbance of the final solution (10 g/ml) was scanned in the range 400-200 nm against 1% v/v H2SO4 in methanol as blank. The scan for determination of max of Levamisole Hydrochloride is shown in Figure 2 (a) and Figure 2 (b).

Then, Overlain spectra of mebendazole and levamisole hydrochloride were also carried out. It is shown in Figure 2 (c).

Preparation of calibration curve of Mebendazole and Levamisole Hydrochloride:

Standard solutions of Levamisole Hydrochloride in the concentration range of 3 g/ml to 18 g/ml were obtained by transferring 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 ml of Levamisole Hydrochloride stock solution (100 ppm) to a series of six volumetric flasks of 10 ml and standard solutions of Mebendazole in the concentration range of 2 g/ml to 12 g/ml were obtained by transferring 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 ml of Mebendazole stock solution (100 ppm) to a series of six volumetric flasks of 10 ml. The volume in each volumetric flask was made up of 1% v/v H2SO4 in methanol as solvent. The absorbance of the solutions was measured at both the wavelength 286.0 nm and 245.0 nm against the 1% v/v H2SO4 in methanol as blank and calibration curves were plotted. The results are shown in Tables 3 (a) and 3 (b).

Other optical parameters i.e. Beer’s limit, slope, intercept, and correlation coefficient were calculated from the calibration curve. The parameters ARE reported in Table 1.

Preparation of synthetic API mixture of Mebendazole and Levamisole Hydrochloride:

The synthetic API mixture of Mebendazole and Levamisole Hydrochloride was prepared in ratio of 1:1.5. Accurately weighed 100 mg of Mebendazole and 150 mg of Levamisole Hydrochloride were transferred to a 100 ml volumetric flask, 100 ml of 1% v/v H2SO4 in methanol as solvent was added to it up to quantity sufficient tan sonicated properly. Further 10 ml of prepared solution (1000 ppm) was transferred to a 100 ml volumetric flask. The volume was adjusted to 100 ml with 1% v/v H2SO4 in methanol as solvent. The final standard stock solution contains 100 g per ml of Mebendazole and 150 g per ml of Levamisole Hydrochloride.

The Simultaneous Equation Method:

The stock solution of the synthetic mixture (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 ml) was transferred to six 10 ml volumetric flasks. The volume was made up to 10 ml with 1% v/v H2SO4 in methanol as solvent. The absorbances of these solutions were measured at 286 nm and 245 nm wavelengths.

METHOD VALIDATION

The proposed method has been validated by various parameters: linearity, range, accuracy, precision, limit of detection, limit of quantification, specificity, and robustness[15].

Linearity:

Linearity was observed in the range of 2-12 g/ml for MBZ and 3-18 g/ml for LVM, respectively with an r2 value of greater than 0.995.

Range:

The range of an analytical method is defined as the interval between upper and lower levels (including these levels). It includes working range, linearity range, target range, and 100% concentration.

·         Working range - the working range was found to be 0.441 to 12.0 g/ml & 0.681 to 18.0 g/ml for Mebendazole and Levamisole Hydrochloride respectively.

·         Linearity range - It was found to be equal to 2.0 – 12.0 g/ml & 3.0 – 18.0 g/ml for Mebendazole and Levamisole Hydrochloride respectively.

·         Target range - These were equal to 5.6 g/ml, 7.0 g/ml and 8.4 g/ml for Mebendazole and 8.4 g/ml, 10.5 g/ml and 12.6 g/ml for Levamisole Hydrochloride.

·         Target concentration – It was 7.0 ppm for Mebendazole and 10.5 ppm for levamisole      Hydrochloride. For the mixture, it was 8.75 ppm.

Precision:

Variation in absorbance in intraday ( 3 different times in a day) and in interday (3 consecutive days at the same time) was determined. Three dilutions (2.4:3.6, 3.0:4.5, and 3.6:5.4) were made for this from the stock solution of the mixture of Mebendazole: Levamisole. The repeatability test was also performed by preparing 6 samples of the same concentration (3.0:4.5) from the stock solution. Then the % RSD of these three tests was calculated to determine whether the developed method was precise or not. The low %RSD value suggests that the developed method is precise. The results are reported in Table 2.

Accuracy:

The accuracy of the developed methods was determined by calculating % recovery at three different levels (80%, 100%, and 120%) in pre-analyzed samples using the standard addition method. 2 ml of the pre-analyzed solution (100 g/ml Mebendazole and 150 g/ml Levamisole Hydrochloride) was taken and transferred to 10 ml volumetric flask, volume was made up to the mark with 1% v/v H2SO4 in methanol to get the desired stock solution (20 g/ml of MBZ and 30 g/ml of LVM). Further, 0.75 ml of pre-analyzed stock solution (20 g/ml of MBZ and 30 g/ml of LVM) was taken, and 0.45, 0.75 and 1.05 ml of standard solution of API mixture (20 g/ml MBZ and 30 g/ml LVM) was added and volume was made up to the mark with 1% v/v H2SO4 in methanol as a solvent. The results of recovery studies are reported in Table 2. The % recovery for MBZ and LVM are within 99.108% - 99.443%, assuring that both the developed methods can estimate the drugs successfully in the presence of excipients. The results are reported in Table 2.

LOD and LOQ:

LOD and LOQ were determined from the standard deviations of the responses for six replicate determinations.

The limit of detection (LOD) and limit of quantitation (LOQ) may be expressed as LOD = 3.3(SD/S)

Where, SD= Standard deviation of the response S= Slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte.

LOQ = 10(SD/S)

Where, SD = Standard deviation of the response S = Slope of the calibration curve

The results are reported in Table 2.

Specificity:

The tablet dosage form was made through mebendazole, levamisole, and common excipients. Then 100ppm solution was prepared from it. Further 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 ml of resulted solutions were transferred to a series of six volumetric flasks of 10 ml separately and volume was made up to the mark with 1% v/v H2SO4 in methanol. Then a synthetic mixture was formed by mixing the pure drug of levamisole hydrochloride and mebendazole in 100 mg and 150 mg ratio and made its dilutions as above. The % interference below 0.5% suggests that the excipients have a negligence effect on the effect of active constituents. The results are reported in Table 2.

Robustness:

The mixture having a concentration of 3 µg/ml of mebendazole and 4.5 µg/ml of levamisole hydrochloride was prepared from the standard stock solution. The prepared solution was analyzed as per the proposed method with a small but deliberate change in max. (max. ± 1.5 nm) and scanning speed (slow, medium, and fast). The RSD value less than 2% suggests that the developed method is robust at small variations. The results are reported in Table 2.

Analysis of marketed formulation:

20 tablets were weighed and powdered. A quantity equivalent to 10 mg of Mebendazole and 15 mg of Levamisole Hydrochloride was transferred to a 100 ml volumetric flask and mixed with 70 ml of 1% v/v H2SO4 in methanol as a solvent and the solution was sonicated for 20 minutes thereafter volume was made up to 100 ml with the same solvent to produce the resultant solution of 100 g/ml and 150 g/ml of Mebendazole and Levamisole Hydrochloride. The solution was filtered through Whatman filter paper 40. From the filtrate, 2 ml was transferred to five different 10 ml volumetric flasks, and the volume in each was made up to 10 ml with 1% v/v H2SO4 in methanol as a solvent to produce the resultant solution of 20 g/ml and 30 g/ml of Mebendazole and Levamisole Hydrochloride respectively. Further 1.5 ml was transferred to five different 10 ml volumetric flasks and volume in each was made up to 10 ml with 1% v/v H2SO4 in methanol as a solvent. Absorbances of these solutions were measured at 286.0 nm and 245.0 nm using 1% v/v H2SO4 in methanol as blank. Then it concentration of the active constituent in each tablet was calculated. It should be present between 98-102%.

RESULTS AND DISCUSSION

The standard solutions of MBZ and LVM were scanned separately in the UV range and simultaneous equation methods for MBZ and LVM were recorded. For mebendazole, the λmax was found at 286.0nm, and for levamisole hydrochloride, the λmax was found at 245nm. These two wavelengths can be employed for the determination of MBZ and LVM without any interference from the other drug in their combined dosage form. Standard calibration curves for MBZ and LVM were linear with Correlation coefficients (r2) values in the range of 0.998 and 0.999, respectively at all the selected wavelengths, and the values were average of six readings and statistical data is shown in (Table 1 ). The developed method was found to be precise as the %RSD values for the intermediate precision studies were < 2%. (Table 2) The accuracy of the proposed method was ascertained by recovery studies and the results were expressed as percent recovery and were found in the range of 99.108% – 99.443%. Values of standard deviation and coefficient of variance were satisfactorily low indicating the accuracy of the method. (Table 2) The influence of excipients was studied by mixing two drugs with excipients as per the ratio. LOD and LOQ were found to be 0.145 g/ml 0.441 g/ml for MBZ and 0.225 g/ml and 0.681 g/ml for LVM. Table 2 shows the summary of all validation parameters. The assay results of tablet formulation show drug concentration between 98-102% which shows good agreement with the labeled claim. So it proved that no interference was observed from the presence of excipients in the amounts, which are commonly present in tablet formulation.

REFERENCES

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Figure 2 (a): UV scan for determination of max. of Mebendazole

Figure 2(b): UV scan for determination of max. of Levamisole         Hydrochloride

Figure 2(c): UV scan for simultaneous estimation

Figure 3 (a): Calibration curve for Mebendazole at 286 nm

Figure 3 (b): Calibration curve for Mebendazole at 245 nm

Figure 3 (c): Calibration curve for Levamisole Hydrochloride at 286 nm

Figure 3 (d): Calibration curve for Levamisole Hydrochloride at 245 nm.

Table 1: Regression characteristics for Mebendazole and Levamisole Hydrochloride