INTRODUCTION:

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs^1-8.

Levamisole C₁₁H₁₂N₂S is an antihelminthic drug that was commonly used for the treatment of parasitic, viral, and bacterial infections. It is an antiparasitic agent appears to be tied to its agnositic activity towards the L-subtype nicotinic acetylcholine receptors in nematode muscles. This agonistic action reduces the capacity of the males to control their reproductive muscles and limits their ability to copulate^9-15.

Figure 1: Structure of Levamisole

An attempt has been made to develop and validate a simple, economic, rapid and accurate method. The proposed method was validated according to ICH guidelines^16-21.

The reported simple RP-HPLC method used methanol: Methanol and 0.05% Ortho phosphoric acid (60:40v/v) as a mobile phase. The goal of this study was to develop a method without using buffer in mobile phase, has less run time, and more sensitive compare to developed method for analysis of Levamisole, with extremely low LOD and LOQ values.

MATERIALS AND METHODS:

Levamisole was procured from Yarrow-chem, Mumbai, India. HPLC Grade methanol, Ortho-phosphoric acid obtained from S.D. Fine Chem, Pvt. Ltd. and Laboratory grade Hydrochloric acid, hydrogen peroxide and sodium hydroxide obtained from Loba Chem, Mumbai.

Instrumentation:

Agilent technology HPLC instrument was used for the analysis purpose. Column Agilent C₁₈ (250mm × 4.6 mm). UV-2080 UV-Visible Spectrophotometer Analytical Technologies Pvt. Ltd²².

RESULT AND DISCUSSION:

Chromatographic Conditions:

The chromatographic separation was carried out at room temperature on Agilent C₁₈ (250mm × 4.6 mm) analytical column under RP-HPLC conditions. Mobile phase was Methanol: 0.1% Ortho phosphoric acid (80:20% v/v). The mobile phase was filtered through 0.45 micron membrane filter which was degassed by ultrasonic bath. Flow rate was 0.9 ml/min. The detection Wavelength was 227 nm. The injection volume was 20-µl.

Selection and Preparation of Mobile Phase:

0.1% ortho phosphoric acid is prepare by take a 0.1ml ortho phosphoric acid and dissolved in 100ml water. An accurately measured quantity of 320ml methanol and 80 ml 0.1% ortho phosphoric acid were transferred in a 500 ml volumetric flask followed by proper mixing of resultant solution, then filtered through 0.45membrane filter and sonicated with intermittent shaking for the 15 min in a bath sonicator. And inject the 50 µg/ml standard solution with injection volume 20µl flow rate 0.9 ml/min and Wavelength 227nm. Based on the optimized chromatographic conditions the chromatogram is shown in figure no 2.

Optimized Chromatographic Conditions:

The optimized chromatographic conditions are shown in Table .1

Table 1: Optimized Chromatographic Conditions

Column	Agilent C₁₈ (250mm × 4.6 mm)
Flow rate	0.9 ml/min
Wavelength	227nm
Injection volume	20 µL
Mobile phase	Methanol: 0.1% Ortho phosphoric acid (80:20% v/v)

Figure 2: The chromatogram of method development and optimization

Preparation of Standard Solution:

Accurately weighed 10 mg of Levamisole was transferred to a 10 ml volumetric flask, sufficient amount of Methanol was added to dissolve it and volume was made up to 10 ml. pipette out 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.6 ml and diluted with mobile phase (Methanol: 0.1% Ortho phosphoric acid (80:20% v/v) up to 10 ml to get concentration of 10, 20, 30, 40, and 60 µg/ml for the linearity study.

Method Validation:

The validation is performed According to ICH guidelines.

Linearity:

The Linearity of Analytical procedure is its ability to obtained test result which are directly proportional to the concentration of analyte in the sample. The range of 10-60µg /ml was selected for the linearity of a standard Levamisole. The chromatogram of linearity as show in Figure.3 and standard calibration curve is show in Figure.4 and Result of linearity in Table .3.

Figure 3: Chromatogram of Linearity

Figure 4: Standard calibration curve of Levamisole

Table 3: Results of linearity

Sr No.	Conc	Area I	Area II	Mean	SD	%RSD
1	10	335.29	336.01	335.65	0.51	0.15
2	20	739.94	743.73	741.84	2.68	0.36
3	30	1150.26	1141.85	1146.06	5.95	0.52
4	40	1562.06	1556.27	1559.17	4.09	0.26
5	60	2397.8	2397.05	2397.43	0.53	0.02
r²=0.999	Slope= 41.23	Y-intercept= 83.38		Avg. SD=2.75

System Suitability Test:

The system suitability test is a pharmacopoeial requirement and is carried out to verify whether the analytical system is adequate or not for analysis of drug. The 20µg/ml of Levamisole standard solution was prepared and injected same solution into three replicates the Chromatogram of system suitability test is show in Figure.5. and results of system suitability test in Table 4.

Figure 5: System Suitability Test Chromatogram

Table 4: Result of system suitability

Sr. No	Concentration	Peak area	No. of Theoretical plates	Asymmetric factor
1	20 (µg/ml)	746.08	8096	0.77
2	20 (µg/ml)	745.59	7867	0.77
3	20 (µg/ml)	746.34	8065	0.76
Mean 746.00
S.D. 0.346482
%RSD 0.04644

Accuracy study:

The recovery study was perform to evaluate the developed method was accurate for the analysis of Levamisole. The 80%, 100% and 120% levels of recovery study were selected to perform the recovery study. The result of accuracy study was shown in Table 6 and 7.

Table 6: Result of % Recovery Test

Level of % recovery	Amount of tablet solution taken (µg/ml)	Amount of standard solution add (µg/ml)	Area	Amount found	Amount Recovery	% Recovery
80%	10	8	659.39	18.01	8.01	100.12
80%	10	8	661.42	18.06	8.06	100.75
100%	10	10	742.87	20.04	10.04	100.40
100%	10	10	740.32	19.97	9.97	99.70
120%	10	12	820.11	21.91	11.91	99.25
120%	10	12	821.23	21.94	11.94	99.50

Table: 7 Statistical result of accuracy

Level of % recovery	Mean% recovery	SD	%RSD
80%	100.44	0.45	0.44
100%	100.05	0.49	0.49
120%	99.38	0.18	0.18
Mean	99.95

Precision:

ICH defines the precision of an analytical procedure as the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of the method was demonstrated by intra-day and inter-day studies. The 10µg/ml, 30µg/ml, 60µg/ml standard Levamisole solution was suitably selected for method repeatability.

A) Intra-day Precision:

In the intra-day studies, 2 replicates of 3 standard solutions (10µg/ml, 30µg/ml, 60µg/ml) were analyzed in a same day and percentage RSD was calculated. As show in Table .8.

Table 8: Result of Intra-day Precision

Sr No.	Conc.	Area-I	Area-II	Mean	Amount Found	%Amount Found	SD	%RSD	%RSD mean
1	10	333.6	332.54	333.07	10.10	101.04	0.75	0.23	0.093
2	30	1152.24	1151.71	1151.98	29.96	99.86	0.37	0.03
3	60	2400.74	2401.31	2401.03	60.26	100.43	0.40	0.02

Table 9: Result of Inter-day Precision

Sr No.	Conc.	Area-I	Area-II	Mean	Amount Found	%Amount Found	SD	% RSD	%RSD Mean
1	10	339.83	336.18	338.01	10.22	102.2	2.58	0.76	0.35
2	30	1156.52	1152.91	1154.72	30.03	100.1	2.55	0.22
3	60	2402.09	2404.5	2403.30	60.31	100.5	1.70	0.07

Table 10: Result of robustness

Flow Rate	Sr. no	Conc. (µg/ml)	Area	SD	%RSD	%RSD Mean
(0.8ml/min)	1	20	847.72	4.75	0.56	0.78
(0.8ml/min)	2	20	854.44	4.75	0.56
(1.0ml/min)	1	20	682.67	6.78	1.00
(1.0ml/min)	2	20	673.08	6.78	1.00
Mobile Phase	Sr. no	Conc. (µg/ml)	Area	SD	SD	%RSD Mean
(79:81%v/v)	1	20	739.38	8.34	1.12	0.29
(79:81%v/v)	2	20	751.15	8.34	1.12
(81:19% v/v)	1	20	747.04	0.84	0.11
(81:19% v/v)	2	20	748.23	0.84	0.11
Wavelength (nm)	Sr. no	Conc. (µg/ml)	Area	SD	%RSD	%RSD
(226nm)	1	20	745.39	3.50	0.47	0.28
(226nm)	2	20	740.44	3.50	0.47
(228nm)	1	20	747.96	5.07	0.11
(228nm)	2	20	740.79	5.07	0.11

B) Inter-day Precision:

For the interday variation studies, 2 replicates of 3 standard solutions (10µg/ml, 30µg/ml, 60µg/ml) were analyzed on 2 consecutive days and percentage RSD was calculated. As show in Table 9.

Robustness:

ICH defines the robustness of an analytical method was carried out to confirm the analytical method remains unaffected by small variations in the optimized method parameters, the 20µg/ml of standard solution were injected for a each varied conditions like change in flow rate ± 1 ml/min, change in wavelength ± 1 nm and change in composition of mobile phase ± 1 ml and the chromatograms were recorded, then results were obtained by calculating the %RSD of a peak area for each varied conditions. The results of robustness in Table 10.

Ruggedness

ICH defines the ruggedness or Intermediate precision of a method was assessed to check the effect of change in analyst on analysis, different laboratories, instruments and environmental conditions of Levamisole , the linearity as a method validation parameter was repeated by second /other analyst to verify the ruggedness, and % RSD was calculated. The % RSD was found to be 0.63 which is under accepted criteria; hence the developed method was rugged. As show in Table.11.

Table 11: Result of Ruggedness

Sr No	Conc	Area-I	Area-II	Mean	SD	%RSD
1	30	1963.64	1945.15	1954.40	13.07	0.67
2	40	1986.66	1989.14	1987.90	1.75	0.09
3	60	2227.84	2192.09	2209.97	25.28	1.14
AVG SD = 13.36			AVG %RSD = 0.63

Forced Degradation Study:

Alkaline Degradation:

An accurately weighed quantity of 10 mg Levamisole was transferred to the round bottom flask, then 10 ml of 0.1N NaOH was added into it, afterwards; refluxed at 60^oC for 1hr by using reflux assembly, after 1hr the flask was removed to cool the solution, 500l (0.5 ml) solution was withdrawn and transferred into 10 ml of volumetric flask and the final volume was made up to mark with selected mobile phase, the resultant sample was filtered through 0.45 µ membrane syringe filter. Finally, the sample was injected into the system to record the chromatogram. The percent degradation of drug in alkaline condition was found to be 5.06%.

Figure 6: Chromatogram of base degradation sample

Acid degradation:

An accurately weighed quantity of 10 mg Levamisole was transferred to the round bottom flask, then 10 ml of 0.1N HCl was added into it, afterwards; refluxed at 60^oC for 1hr by using reflux assembly, after 1hr the flask was removed to cool the solution, 500µl (0.5 ml) solution was withdrawn and transferred into 10 ml of volumetric flask and the final volume was made up to mark with selected mobile phase, the resultant sample was filtered through 0.45µ membrane syringe filter. Finally, the sample was injected into the system to record the chromatogram. The degradation of drug in acidic condition was found to be 4.71%.

Figure.7 Chromatogram of acid degradation sample

Peroxide degradation:

An accurately weighed quantity of 10 mg Levamisole was transferred to the round bottom flask, 10 ml of 3% H₂O₂ was added, afterwards; refluxed at 60^oC for 1hr. after 1hr flask was removed to cool the solution, 500 µl (0.5 ml) of solution was withdraw and transferred to a 10 ml volumetric flask and the final volume was adjusted up to mark with mobile phase, the resultant sample was filtered through 0.45µ membrane syringe filter, and inject it. The degradation of drug in oxidative condition was found to be 4.04%.

Figure 8: Chromatogram of peroxide degradation sample

Thermal degradation:

The sample was heated in loss on drying oven at 60°C in Petri plate for 24 Hour. An accurately weighed quantity of 10 mg sample was transferred into 10 ml of volumetric flask and 5 ml of mobile phase was added to it, the resultant solution was sonicated for 25 min with intermittent shaking and diluted up to the mark with mobile phase, then it was allowed to settle for 15 min, the 100µl of supernatant solution was diluted up to 10 ml by mobile phase, then filtered through 0.45µ membrane syringe filter-media and injected into the system to obtain the chromatogram Figure.9. The degradation of drug in thermal condition was found to be 5.45%.

Figure 9: Chromatogram of thermal degradation sample

Table 12: Result of forced degradation study

Sr. No.	Stress Condition	% Degradation
1	Alkaline condition	5.06%
2	Acidic condition	4.71%
3	Oxidative condition	4.04%
4	Thermal condition	5.45%

Table 13: Results of validation

Sr. No.	Parameter	Result	Acceptance criteria
1	Linearity	10-60µɡ/mL, R² = 0.999	R²≥0.999
2	System suitability test	T. plates=8009, A. factor=0.76	T.Plates≥2000, A. factor<1.75
3	Accuracy	99.95	98-102%
4	Intraday precision	0.09%	%RSD < 2%
5	Intra-day precision	0.35%	%RSD < 2%
6	Specificity	No Interference	No Interference
7	Robustness	Change in wavelength: 0.28%, Change in flow rate:0.78%, Change in composition: 0.29%	%RSD < 2%
8	Ruggedness	0.63	%RSD < 2%
9	LOD and LOQ	0.22 and 0.66	LOQ is three times of LOD

The forced degradation study was carried out in acid, base, peroxide and thermal stress condition. In alkaline condition 5.06% drug was degraded. In acidic condition 4.71% drug was degraded and in oxidative drug was 4.04% degraded. In thermal degradation, 5.45% of drug was degraded at 60⁰C.

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Received on 15.07.2021 Modified on 21.11.2021

Asian J. Pharm. Ana. 2022; 12(2):99-104.

DOI: 10.52711/2231-5675.2022.00018