Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/15599
Title: Simultaneous Determination of Active Ingredients in Fixed Dose Pharmaceutical Combinations Using RP-HPLC
Authors: Ali, Amir
Keywords: Physical Sciences
Chemistry
Issue Date: 2020
Publisher: University of the Punjab , Lahore
Abstract: In the present dissertation, efforts were applied to determine vitamins, antihistamine and ophthalmic drugs in fixed dose combinations using simple, sensitive and economic isocratic stability indicating HPLC methods. Four stability indicating HPLC methods have been developed for the following fixed dose combinations i.e. (Naphazoline Hydrochloride and Antazoline Hydrochloride), (Paracetamol, thiamine and Pyridoxal phosphate), (Chlorpheniramine Maleate and Prednisolone acetate) and (Diazepam and Pyridoxine). Two LC/MS-MS methods have been developed for the estimation of following antibiotics in human blood plasma i.e. Enrofloxacin and Florfenicol. In the first combination, a newly developed method based on ultrahigh performance liquid chromatography (UHPLC) was optimized for the simultaneous determination of antazoline hydrochloride (ANZ) and naphazoline hydrochloride (NFZ) in ophthalmic formulations. Isocratic separation of ANZ and NFZ was performed at 40 °C with an ACE Excel 2 C18- PFP column (2 μm, 2.1 × 100 mm) at a flow rate of 0.6 mL min–1 whereas the mobile phase consisted of acetonitri- le/phosphate buffer (60:40, v/v, pH 3.0) containing 0.5% triethylamine. Both analytes were detected at a wavelength of 285 nm and the injection volume was 1.0 μL. The overall run time per sample was 4.5 min with retention time of 0.92 and 1.86 min for NFZ and ANZ, respectively. The calibration curve was linear from 0.500–100 μg mL–1 for ANZ and NFZ with a le repeatability and reproducibility (expressed as relative standard de- viation) were lower than 1.28 and 2.14%, respectively. In comparison with high-performance liquid chromatography (HPLC), the developed UHPLC method had remarkable advantages over HPLC as the run time was significantly redu- ced by 3.4-fold with a 5-fold decreased solvent consumption. Forced degradation studies indicated a complete separa- tion of the analytes in the presence of their degradation products providing high degree of method specificity. The pro- posed UHPLC method was demonstrated to be simple and rapid for the determination of ANZ and NFZ in commer- cially available ophthalmic formulations providing recoveries between 99.6 and 100.4%. In the second combination, a precise, accurate and reliable liquid chromatographic method was developed for simultaneous determination of paracetamol, thiamine, and pyridoxal phosphate in pharmaceutical formulations. The separation of the analytes was carried out with Agilent Poroshell C18 column. The mixture of ammonium phosphate buffer (pH = 3.0), acetonitrile and methanol at the ratio of 86:7:7 (V/V/V) was used as the mobile phase pumped at a flow rate of 1.8 mL min-1 . The detection of all three components, impurities and degradation products was performed at the selected wavelength of 270 nm. The developed method was validated in terms of linearity, specificity, precision, accuracy, LOD and LOQ as per ICH guidelines. The linearity of the developed method was found in the range 17.5-30 µg mL-1 for thiamine, 35-60 µg mL-1 for pyridoxal phosphate and 87.5-150 µg mL-1 for paracetamol. The coefficient of determination was found to be ≥ 0.9981 for all three analytes. The proposed HPLC method was found simple, reliable, precise and accurate for the routine analysis of paracetamol, thiamine and pyridoxal phosphate in tablet formulations. Whereas the complete separation of analytes in the presence of degradation products is justification of method stability. In the third combination, a simple, rapid, sensitive, accurate and precise method for simultaneous determination of chlorpheniramine maleate (CHRM) and prednisolone (PRED) in injection samples by HPLC coupled with UV-Vis detector. The chromatographic separation was accomplished employing isocratic mode and the mobile phase comprised of acetonitrile and phosphate buffer (50:50, v/v, 30 ˚C), adjusted to pH 3.0. The flow rate used was 1.0 mL/min on Thermo Hypersil ODS C18 column (5μm, 4.6 x 250 mm) and injection of samples was 20 µL. The analysis of CHRM and PRED was performed at a wavelength of 254 nm. The runtime for analysis was 12.5 min and retention times of CHRM and PRED were found to be 2.81 and 5.07 min, respectively. The calibration graph showed linearity over the concentration range 10-70 µg/mL for CHRM and 20-140 µg/mL for PRED with coefficient of determination (R2 ) ≥ 0.9986. Repeatability and reproducibility (expressed as RSD %) were lower than 1.72 and 1.47 %, respectively. The proposed HPLC method was demonstrated to be simple and rapid for the determination of CHRM and PRED in injection formulation; providing recoveries between 101.6-102.3 % whereas complete separation of degradation products, from analyte under investigation, provided the specificity of proposed HPLC method. In the fourth combination, stability-indicating HPLC assay method was established for the estimation of Diazepam and Pyridoxine in pharmaceutical formulation. Water C 8 (250x4.6mm, 5μm) column was employed to separate analytes and degradation products, using Phosphatic buffer (pH=3.0) and ACN (30:70) as a mobile phase, pumped at a flow rate of 1.0 mL/min. The detection of API and degradation product was carried out at a wavelength of 223 nm. The retention times of DZP and PY were found 5.02 and 2.15 min respectively. The validation of proposed method was performed in accordance to ICH guidelines. The calibration graphs showed that proposed method was linear within range of 12-84 μg/mL for DZP and 20-140 μg/mL for PY. LOD and LOQ values were found 0.122 and 0.287 respectively for DZP while 0.264 and 0.431 for PY. The recovery was found between the ranges 99.80-100.64 % and 100.46–100.91 % for DZP and PY respectively by proposed HPLC method RSD values were found less than 1.72 and 1.43 for repeatability and reproducibility respectively, for DZP while below than 1.32 for repeatability and 1.42 for reproducibility assays of PY. Forced degradation studies established that the proposed method was specific for the estimation of DZP and PY. The developed HPLC method was found simple, accurate and precise for routine analysis of DZP and PY in commercial pharmaceutical formulations. An accurate LC/MS-MS method was established for the estimation of enrofloxacin in human plasma. The separation of components was achieved by C8 HPLC column (250 x 4.6mm, 5μm Waters Corp., Milford, MA, USA) using formic acid (2 %) in H2O, MeOH and ACN (56:7:37, v/v) as mobile phase, pumped at a flow rate of 1.5 mL/min. The mass spectrometer with electrospray ionization in a multiple reaction monitoring (MRM) mode was used. The analyte enrofloxacin and ciprofloxacin (Internal standard) showed high sensitivity in positive ion detection mode. The validation of developed method was performed as per ICH guidelines. The developed method was found linear within concentration range of 0.25-100.0ng/mL with coefficient of determination (R2 ) ≥ 0.9975. The values of intra-day precision for low, medium and high QC samples were found 7.85, 3.56 and 2.55 respectively, while for inter-day precision were 7.62, 0.41 and 0.47 respectively, with accuracy ranged from 84.0 to 112.4 %. The total run time for analysis of enrofloxacin was found less than 3.0 min. The developed method was demonstrated for the estimation of enrofloxacin in human plasma. An accurate LC/MS-MS method was established for the estimation of florfenicol in human plasma. A C8 HPLC column (250 x 4.6mm, 5μm Waters Corp., Milford, MA, USA) was used to separate the components by isocratic elution mode. The mixture of ACN and water (75:25 v/v) was used to elute components at flow rate of 1.5mL/min. The developed method was found linear within range of 0.25-100.0ng/mL with coefficient of determination (R2 ) ≥ 0.9965. The values of intra-day precision for low, med and high QC samples were found 4.35, 2.87 and 3.48 respectively, while for inter-day precision were 7.06, 15.56 and 21.67 respectively, with accuracy ranged from 78.0 to 108.9 %. The total run time for analysis was found less than 3.0 min. The retention time of florfenicol was found 1.89 min. The value of LLOQ was found 0.75 ng/mL for florfenicol. The proposed method was demonstrated for the estimation of florfenicol in human plasma.
Gov't Doc #: 20724
URI: http://prr.hec.gov.pk/jspui/handle/123456789/15599
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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