Validated Analytical Method Development for the Quantification of Multicomponent Drugs with Special Emphasis on Herbal and Synthetic Drug Formulations

The present work involved the development of accurate, precise, simple and rapid Spectroscopic (UV) and Chromatographic (HPLC, HPTLC) methods for simultaneous estimation of the drugs in synthetic and herbal multicomponent formulations containing: - Losartan potassium and Chlorthalidone. - Acebrofylline and Acetylcysteine. - Atovaquone and Proguanil. - Lawsone and p-phenylenediamine. - L- dopa in Zandopa herbal powder. This thesis invoves different chapter providing valuable information about the related topic studied detailed in this thesis. Chapter first deals with the brief introduction about modern analytical techniques like hplc, hptlc, and uv-spectroscopic method and gives brief information about the methods and techniques for the estimation of the multicomponent drug analysis. In this chapter also involves detailed about analytical method developments, analytical method validation, herbal analysis and forced degradation study. The Introduction of present work covers optimum information about the project name entitled here. The objectives of the study were to develop and validate methods for the estimation of drugs in multicomponents drug formulations by using HPLC, HPTLC, and UV Spectrophotometry was explained. In next chapter of this thesis deals with review of literature for the analytical methods available for the estimation of the selected drug formulation either present in single or in the combination with the other drug products. In Fourth chapter discuss about the scope and planning for achieving the objectives of the study explain here. The material and methods chapter described with step by step procedure performed for the optimizing condition for the development of method on HPLC, HPTLC and UV Spectrophotometry methods and validation of the developed method were performed in accordance with ICH guideline and the methods also described the stability indicating assay method carried out by subjecting sample to the stress condition like acid, alkali, peroxide, neutral, thermal and UV light. The optimized chromatographic and spectrophotometric conditions are presented along with data table, graphs, and typical chromatogram were discuss. In the next chapter of this thesis deals with result and discussion are presented in series of tables and figures which includes all the trials chromatograms, the calibration curve, validation data, degradation study data, and herbal analysis of drugs by using modern analytical instruments like HPLC, HPTLC and UV Spectroscopic methods. The over all of the developed methods were summarized below. In the HPLC method developed for losartan potassium and chlorthalidone in tablet (CTD-L) formulations, isocratic mobile phase was found to be unsuitable. Thus gradient conditions were chosen which brought about the elution of losartan potassium and chlorthalidone. The optimized chromatographic conditions were- Injection volume : 10μl. Analytical column : Agilent XDB, C18, 150 × 4.6 mm, 5μ. Detection : 254. Mobile phase : Buffer: acetonitrile in (70:30), pH3.5. The adopted Isocratic system and chromatographic conditions resolved the losartan potassium and chlorthalidone with retention times of 4.63 and 2.17 min respectively. The method was linear in the range of 12.5 – 75 μg/ml for losartan potassium and 1.56 – 9.37 μg/ml for chlorthalidone. The method was validated for system suitability, accuracy, precision, linearity, range, limit of detection (LOD), limit of quantification (LOQ), specificity and robustness The accuracy and precision studies gave very low values of % relative error and % relative standard deviation. Likewise, the HPLC method developed for atovaquone and proguanil in tablet (MALARONE) formulations, isocratic mobile phase was found to be unsuitable. Thus gradient conditions were chosen which brought about the elution of atovaquone and proguanil. The following gradient system was optimized-Mention the mobile phase for gradient elution. The HPLC method was linear from 25 – 150 μg/ml for acebrofylline and 150 – 900 μg/ml for acetylcysteine. The method was validated for system suitability, accuracy, precision, linearity, range, limit of detection(LOD), limit of quantification (LOQ), specificity and robustness with good results. Stability indicating assay is a method that is employed for the analysis of stability samples in pharmaceutical industry. When developing SIMs, forced degradation studies are undertaken particularly when little information is available about potential degradation products. These studies also provide information about the degradation pathway and degradation products that could form during storage. Forced degradation studies help facilitate pharmaceutical development as well as in cases such as formulation development, manufacturing and packaging in which knowledge of chemical behavior can be used to improve a drug product. With this view, forced degradation studies were carried out for the multicomponent combinations of Losartan-Chlorthalidone, Atovaquone-Proguanil and Acebrophylline-Acetylcysteine. The degradation studies were carried out under acidic, alkaline, oxidative, and dry heat (thermal) and photolytic conditions. The degraded samples and standard drug were subjected to chromatographic separation and the conditions required for resolving the drug peak from any potential degradation products were optimized. During forced degradation studies, Losartan (11.16%)and Atovaquone (11.3%) was found to degrade under peroxide degradation conditions and other drug shows % degradation below 10% which is acceptable range. It can be concluded that the stability indicating HPLC method developed for the multicomponent combinations of Losartan-Chlorthalidone, Atovaquone-Proguanil and Acebrophylline-Acetylcysteine is capable of discriminating between the drug and the degradation products. Simultaneous equation method for estimation of drugs in combination is one of the effective methods of multicomponent. Hence UV spectrophotometric methods were developed and validated for simultaneous estimation of Losartan-Chlorthalidone, Atovaquone-Proguanil and Acebrophylline-Acetylcysteine from multicomponent formulations. The method developed for simultaneous estimation of losartan potassium and chlorthalidone showed absorbance maxima at 235nm and 215nm for losartan potassium and chlorthalidone respectively. The developed method was validated as per ICH Guidelines. The method was found to be accurate, precise, specific, robust and linear in the range of 6-16 for losartan and 0.75 - 2 μg /ml for chlorthalidone. The method developed for simultaneous estimation of Atovaquone and Proguanil showed absorbance maxima at 279nm and 254nm for Atovaquone and Proguanil respectively. The developed method was validated as per ICH Guidelines. The method was found to be accurate, precise, specific, robust and linear in the range of 7.5- 20 for Atovaquone and 3 - 8 μg /ml for Proguanil. The method developed for simultaneous estimation of Acetylcysteine and Acebrofylline showed absorbance maxima at 220nm and 273nm for Acetylcysteine and Acebrofylline respectively. The developed method was validated as per ICH Guidelines. The method was found to be accurate, precise, specific, robust and linear in the range of 6 - 36 for Acetylcysteine and 1 - 6 μg /ml for Acebrofylline. The HPTLC method for estimation of Acetylcysteine and acebrofylline made use of unmodified silica plates and solvent system of chloroform : tolune: methanol (6:2:2 V/V/V). The HPTLC method gave linearity for 300 to 1800 ng/spot of the applied quantity for Acetylcysteine and 50 to 300 ng/spot for Acebrofylline. This method gave good results in the validations studies. The HPTLC method for estimation of Atovaquone and Proguanil made use of unmodified silica plates and solvent system of THF : water (6 : 4 V/V). The HPTLC method gave linearity for 500 to 3000 ng/spot of the applied quantity for Atovaquone and 200 to 1200 ng/spot for Proguanil. This method also gave good results in the validations studies. The HPTLC method for estimation of Losartan and Chlorthalidone made use of unmodified silica plates and solvent system of Tolune :chloroform: methanol: ammonia (2.3: 5.0: 2.0: 0.2 V/V/V/V). The HPTLC method gave linearity for 200 to 1500 ng/spot of the applied quantity for Losartan and 31.25 to 187.5 ng/spot for Chlorthalidone. This method also gave good results in the validations studies. The HPTLC method for estimation of Lawsone and Paraphenylenediamine made use of unmodified silica plates and solvent system of n-butanol: acetic acid: water (7: 0.1: 3 V/V/V). The HPTLC method gave linearity for 10 to 70 ng/spot of the applied quantity for Lawsone and 100 to 700 ng/spot for Paraphenylenediamine. The HPTLC method for estimation of L-dopa made use of unmodified silica plates and solvent system of n-butanol n-butanol:water:acetic acid(4:1:1v/v/v). The HPTLC method gave linearity for 100-500ng/spot for L-dopa. This method also gave good results in validation. All analytical methods summarized above were successfully developed and validated HPLC, HPTLC, and UV Spectrophotometric methods are better, improved and innovative, less expensive and time saving as well as sensitive, specific, linear, precise, and accurate, for the quantification of both the herbal and synthetic multicomponent formulations can be used in the routine quality control of the above mentioned multicomponent formulations