Development and Validation for HPLC Method of Assay of Lvermectin and Clorsulon in Combined Pharmaceutical Dosage Form

Stability indicating-HPLC method has been developed for simultaneous estimation of Ivermectin and Clorsulon in their combined dosage form. For RP-HPLC method, all the standard and sample solutions were prepared in methanol. A RPHPLC method has been developed and subsequently validated for simultaneous estimation of Ivermectin and Clorsulon in their combination product. The proposed RP-HPLC method utilizes a Thermo BDS C-18 (15cm x 4.6mm, 5 μm) column, mobile phase consisting of acetonitrile, methanol and purified water in the proportion of 60: 30:10 (v/v/v), and UV detection at 245 nm. The described method was linear over a range of 10-40μg/ml with a correlation coefficient (r 2 ) of 0.9998 for Ivermectin and a range of 100-400μg/ml with a correlation coefficient (r 2 ) of 0.9998 for Clorsulon. Validations of the proposed method were carried out for its accuracy, precision, linearity and range, specificity, LOD and LOQ according to ICH guidelines. A stability-indicating study was also carried out and indicated that this method can also be used for purity and degradation evaluation of these formulations that occurred due to temperature, humidity and time. the method has been successfully applied for the analysis of drugs in formulation.


Introduction
Ivermectin (IVM) is macrocyclic lactone that has been known as a potent, effective and safe antiparasitic drug for 20 years [1]. It is widely used as an antiparasitic agent in domestic animals and is considered the drug of choice for lymphatic filariasis and river blindness (onchocerciasis) in humans [2]. IVM is a member of the Avermectins; this group includes natural compounds produced by fermentation of the soil-dwelling actinomycete Streptomyces avermitilis. IVM, a semi-synthetic derivative of avermectin B1, consists of an 80:20 mixtures of the equipotent homologous 22, 23 dehydro B1a and B1b [3].
Literature survey reveals a few spectrophotometric and chromatographic methods for the estimation of both drugs as a single component and in combination with other drug [6]. However, no method has been reported for analysis of these drugs in combined dosage form. there is official method for simultaneous estimation of the two drugs in their combined form stated in, United states [7], British [8] or European Pharmacopeias [9].
Ivermectin is a white to yellowish-white, nonhygroscopic, crystalline powder with a melting point of about 155°C. It is insoluble in water but is freely soluble in methanol and soluble in 95% ethanol [10]. On livestock ivermectin is effective against the major parasitic roundworms: gastrointestinal (e.g. Haemonchus spp, Cooperia spp, Ostertagia spp and Trichostrongylus spp) and pulmonary (e.g. Dictyocaulus spp). It is also effecive against most mites and lice species, and against numerous myiases (e.g. those caused by screwworm flies, bot flies and warble flies) usually regardless of the delivery form (pour-on, injectable, drench or feed additive).
Clorsulon is a substance belonging to the benzenensulphonamide family which is used for the treatment and control of adult flukes. Veterinary medicinal products containing clorsulon are currently marketed in the EU for the treatment of cattle. They are available as injectable formulations to be administered subcutaneously (recommended dose 2 mg/kg) or by the oral route (recommended dose 7 mg/kg). Clorsulon is frequently used in association with Ivermectin [11]. Clorsulon is frequently used in association with Ivermectin. Clorsulon was previously assessed by the CVMP and a toxicological ADI of 0.002 mg/kg body weight, i.e. 0.120 mg/person was established.
In susceptible flukes, Clorsulon inhibits the glycolytic enzymes 3-phosphoglycerate kinase and phosphoglyceromutase, thereby blocking the Emden-Myerhof glycolytic pathway. The fluke is deprived of its main metabolic energy source and dies.
Clorsulon is approved for use in the treatment of immature and adult forms of Fasciola hepatica (Liver fluke) in cattle. It is not effective against immature flukes less than 8 weeks old. It also has activity against Fasciola gigantica. Although not approved, the drug has been used in practice in various other species (e.g., sheep, llamas). It has activity against F. magna in sheep, but is not completely effective in eradicating the organism after a single dose, thereby severely limiting its clinical usefulness against this parasite. Clorsulon is also not effective against the rumen fluke (Paramphistomum). Therefore, the objective of present communication is to develop simple, rapid and precise spectrophotometric method for the estimation of Ivermectin and clorsulon in combined pharmaceutical dosage form. Liquid Chromatographic Conditions Chromatographic conditions were obtained using a stainless steel column (Thermo BDS C-18,150mm x 4.6mm 5.0mµ), which was maintained at ambient temperature. The analytical wavelength was set at 245 nm and samples of 10. µl were injected to HPLC system. The mobile phase consisting of a mixture of Acetonitrile: Methanol: Purified Water in the ratio (60:30:10), at a flow rate of 1ml/min. The mobile phase was filtered through 0.45mµ filter and degassed for 10 minutes by sonication. Method Validation Method validation is the process of proving that an analytical method is acceptable for its intended purpose [12]. For pharmaceutical methods, guidelines from the United States Pharmacopeia (USP), International Conference on Harmonization (ICH), and the Food and Drug Administration (FDA) provide a framework for performing such validations. In general, methods for regulatory submission must include studies on specificity, linearity, accuracy, precision, range, detection limit, quantitation limit, and robustness [13].

Expermental
Procedure System Suitability Test From the standard working solution, the concentration was prepared having 20 µg/ml of Ivermectin and 200 µg/ml of Clorsulon. The system suitability test was performed from Five replicate injections of standard working solution.
Linearity In order to study linearity of the response, a series of Seven concentrating levels from 50% -200% of assay analytes concentrations were prepared from stock solutions. The linearity performed was used for the determination of limits of quantification and detection.
From standard stock solution having the concentration 200 µg/ml of Ivermectin and 2000 µg/ml of Clorsulon.
Transfer accurately the specified volume into each of 100ml volumetric flask to obtain the required working standard concentrations explained as follows: Clorsulon Linearity 100 µg /ml: 5 ml was transferred from 2000 µg/ml standard stock solution to 100-ml volumetric flask, then made up to volume with Methanol (50%).
Statistical Analysis and Calculation Formula Used Linearity data should be evaluated using appropriate statistical methods.
A simple regression line of the detector response versus the sample concentration is the most common means of evaluation. Regulatory agencies require the submission of the correlation coefficient, y-intercept, slope of the regression line, and the residual sum of squares for linearity evaluation.
For the current study statistics data analysis (Stata Software) is used (Texas, USA). Formula: Where: At = Area of the Test As = Area of the Standard Cs = Concentration of the Standard Ct = Concentration of the Test Limit of Detection The LOD is the smallest amount of analyte that can be detected, but not necessarily quantitated using a given method. This parameter is important in the use of limit tests as it sets the level below which the method cannot function.
There are several means of calculating LOD for HPLC methods. The most common approach is to determine the sample amount that provides a signal-to-noise ratio of 3:1. Many chromatographic data acquisition systems provide integrated functions to determine the signal-to-noise ratio that are easily employed by analyst.
An alternative to the signal-to-noise approach is to estimate LOD based on the standard deviation of response. For this calculation, LOD = 3.3(SD/S), where SD is the standard deviation of the response based on the standard deviation of the blank, the residual standard deviation of the regression line, and the standard deviation of the y-intercepts of the regression line, and S is the slope of the calibration curve.
Root MSE (SD) = the standard deviation of the yintercepts of the regression line Limit of Quantification The LOQ is the lowest level that an analyte can be quantitated with any degree of certainty.
The LOQ can be determined by a signal-to-noise ratio of 10:1, or approximated by multiplying the LOD by 3.3. As with LOD, this function is easily obtained from current dataacquisition software. Similarly, LOQ can be estimated by the equation LOQ = 10 (SD/S) Root MSE (SD) = the standard deviation of the yintercepts of the regression Specificity Specificity is the ability of a method to discriminate between the analyte (s) of interest and other components that are present in the sample.
Placebo of the Clorsulon and Ivermectin Injection, equivalent to the sample weight was taken and solution prepared similarly to the sample solution. The solution was analyzed as per the proposed method. Sample solution was also analyzed as per the proposed method. No interference from placebo was observed at the retention time of the drugs peaks. The absence of a peak eluting at the retention time of the active ingredient is sufficient to demonstrate specificity for excipients.
Accuracy Accuracy is the closeness in agreement between the accepted true value or a reference value and the measured result obtained. Accuracy studies are usually evaluated by determining the recovery of a spiked sample of the analyte into the matrix of the sample to be analyzed.
The accuracy of the assay method was evaluated with the recovery of the standards from excipients. Three different quantities (low, medium and high i.e. 80%, 100% and 120%) of the authentic standards were added to the placebo. Sample solutions are prepared in triplicate for each spike level as described in the test preparation.
Test Precision Precision is a measure of the ability of the method to generate reproducible results. For the precision study, precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) in triplicate. Repeatability refers to the use of the analytical procedure over a short period of time that was evaluated by assaying of six determinations at 100% of the test concentrations during the same day prepared in the manner described above in the Assay working Solution Page No (11). Intermediate precision was assessed by comparing the assay of six determinations at 100% of the test concentrations on different days (3 days) prepared in the same manner for repeatability.
Robustness Robustness is a measure of the performance of a method when small, deliberate changes are made to the method conditions. The intent of this validation parameter is to identify which, if any, of the method conditions are the most critical to the successful performance of the method.
The Robustness was determined by injecting triplicate injections of standard and by assaying of six determinations at 100% of the test concentrations of the same Ivermectin plus Batch used in the precision Study.
Robustness of the method was checked by varying the instrumental conditions such as flow rate, Organic content in mobile phase ratio, wavelength of detection.

Results and Discussion
System Suitability

Limit of Detection Calculations Ivermectin Component
The method based on the residual standard deviation of a regression line and slope.
The limit of detection and the limit of quantification of the drug were calculated using the following equations as per ICH guidelines. Root MSE is the standard deviation of the error term, and is the square root of the Mean Square Residual (or Error) = SD =3620. 8 The limit of detection (LOD) and the limit of quantification (LOQ)of the drug were established by evaluating the minimum level at which the analyte could be readily detected and quantified. LOD = 3.3 × (3620.8/19600.34) = 0.61 µg/ ml LOQ = 10 × (3620.8/19600.34) = 1.80 µg/ ml Clorsulon Component The method based on the residual standard deviation of a regression line and slope.
The limit of detection and the limit of quantification of the drug were calculated using the following equations as per ICH guidelines. The standard deviation of the response can be determined based on the standard deviation of y-intercepts of regression lines.
Root MSE is the standard deviation of the error term, and is the square root of the Mean Square Residual (or Error) = SD =17180 The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were established by evaluating the minimum level at which the analyte could be readily detected and quantified. LOD = 3.3 × (17180 / 9197.802) = 6.16 µg/ ml LOQ = 10 × (17180 / 9197.802) = 18.68 µg/ ml Specificity  Chromatogram of Placebo and Sample for Specificity Figure 5 shows a Placebo chromatogram demonstrating the absence of a peak for the main component for an injection of a mixture of the excipients.

Discussion
In this work an analytical HPLC method for simultaneous determination of Ivermectin plus injection (Ivermectin + Clorsulon) was developed and validated. The basic chromatographic conditions were designed to be simple and easy to use and reproduce and were selected after testing the different conditions that affect HPLC analysis, for example column, aqueous and organic components of the mobile phase, proportion of mobile phase components, detection wavelength, diluents and concentration of analyte.
To obtain better separation with good resolution these Chromatographic conditions were optimized using a stainless steel column (Thermo BDS C 18,150mm x 4.6mm 5.0mµ), which was maintained at Ambient Temperature. The analytical wavelength was set at 245 nm and samples of 10. µl were injected to HPLC system. The mobile phase consisting of a mixture of Acetonitrile: Methanol: Purified Water in the ratio (60:30:10), at a flow rate of 1ml/min. The mobile phase was filtered through 0.45mµ filter and degassed for 10 minutes by sonication.
To determine linearity a calibration graph was obtained by plotting Ivermectin + Clorsulon injection concentration against peak area. The method was linear in the range of (10 -40 µg/ml) and (100 -400 µg/ml) for Ivermectin and Clorsulon concentrations respectively with a correlation coefficient 0.9998 for Ivermectin and 0.9998 for Clorsulon for more detail refer to tables 3 to 6 and figures 3&4.
To determent the Specificity which is the ability of a method to discriminate between the analyte (s) of interest and other components that are present in the sample. The method shows no interference from placebo was observed at the retention time of the drugs peaks. (See Figure 5 and 6.
The accuracy of the method was assessed by determination of recovery for three concentrations covering the range of the method. The amount of Ivermectin and Clorsulon were recovered in the presence of placebo interference, were calculated. The mean recovery of Ivermectin and Clorsulon were 99.43% and 99.92% respectively which is satisfactory and result was shown in Table 7 and 8.
Precision of the method was done; the% RSD for repeatability (Intra-day precision) were 0.34% and 0.59% for Ivermectin and Clorsulon respectively. The% RSD for intermediate precision were 0.65% and 1.0% for Clorsulon and Ivermectin respectively, Tables 11 to 14 explain the results of the inter-day precision test (intermediate precision). The method was found to be precise Since the RSD% is less than 2.0%.
The robustness of the method was assessed by assaying test solutions under different analytical conditions deliberately changed from the original conditions such as wavelength, flow rate and mobile phase ratio. Assay and system suitability parameters of changed method conditions compared with the original method proves that the analytical method remained unaffected by slight but deliberate changes in the analytical conditions therefore the method is robust. See the results in tables 15, 16, 17, 18 and 19.
A stability-indicating ability of the method is studied by a deliberate degradation through exposure of Ivermectin plus (Clorsulon 100 + Ivermectin 10 mg) to acid hydrolysis, base hydrolysis, photo degradation (U V), heat and Oxidation. The acid and base hydrolysis are significantly affect the two drugs and the resulting degradants are very remarkable in the chromatograms while the other degrading substances show no or very slight influence on the two drugs for details refer to Table 20.
This furnished evidence that the method is suitable for its intended purpose.

Conclusion
The intensive approach described in this manuscript was used to develop and validate a liquid chromatographic analytical method that can be used for simultaneous determination of Ivermectin and Clorsulon in a pharmaceutical dosage form (injection). This HPLC -method for simultaneous estimation of the combined drug was successfully developed and validated for its intended purpose. The method clearly proves to be specific, linear, precise, accurate, robust and Stability-Indicating.