Browsing by Author "Alaama, Mohamed"

Generic formulation is getting popular world-wide. Demand for reliable analytical and bio analytical technique is getting more attention from the researchers around the globe. A total analytical and bio analytical solution for generic formulations are reported in this study. Analytical and bioanalytical methods were developed and validated for the determination of antihypertensive drugs (Amlodipine (AMLO) and perindopril (PER)) and antidiabetics drug (gliclazide (GLZ)) in pharmaceutical formulations and human plasma using HPLC and LC-MSMS. Firstly, new sensitive and economic analytical method was developed and validated for the analysis of AMLO in tablet dosage form using High Performance Liquid Chromatography (HPLC) with UV detector. The degradation profile of AMLO was identified and the degradation diagram was created. In second part, an HPLC with Diode Array Detector (DAD) was developed and validated for the determination of AMLO in human plasma with cetirizine (CTZ) as internal standard. Sample preparation was optimized using DOE (Design of Experiment). Three different parameters namely solvent type, solvent volume and pH were monitored at different levels for the optimization of sample preparation that would produce best recovery of the analytes. Chromatographic separation of the analytes was obtained using Phenomenix C18 (150 × 4.5 mm, 5 µm) column. The third method was the analysis of GLZ in tablet dosage form using HPLC instrument with modern rapid resolution column. Samples were injected to C18 Waters Xselect HSS C18 (2.1 × 100 mm, 2.5 µm) column. The method showed high recovery, precision and repeatability. The degradation products were identified and some products were reported for the first time. In fourth part, we developed and validated new LC-MSMS method for the determination of GLZ in rats’ plasma using CTZ as internal standard. The method was developed using Aquity Waters H Class UPLC connected to Xevo TQD MS detector. The total run time was 5 min with a flow rate of 0.4 ml/min and a total volume of 2ml of solvent consumption making this method highly economic. The method was used to study the interaction between amlodipine and gliclazide when they are administered together. It was found that the co-administration of AMLO and GLZ led to the prolonged elimination of GLZ from rats’ plasm. This is the first report on the pharmacokinetic interaction between AMLO and GLZ. The method was extended for the simultaneous determination of AMLO and GLZ in human plasma with satisfactory results. Fifth part was to develop and validate an LC-MSMS method with Multiple Reaction Monitoring (MRM) mode for the analysis of AMLO and PER in combined tablet dosage form. Ultra-Performance Liquid Chromatography with Mass Spectrometer Detector (UPLC-MSMS) was used for the method presented in this study as the samples were injected into Waters BEH C18, 1.7 um column. The detection was performed using Xevo triple quadrupole Mass Spectrometer (TQD-MSMS) detector which was operated at MRM and electrospray positive mode. The chromatographic and detection conditions were optimized using design of experiment for the highest response. Last part was to develop and validate LC-MSMS method for the determination of AMLO and PER simultaneously in human plasma. All the methods were validated following standard guideline such as International Conference of Harmonization (ICH) for dosage forms and Centre for Drug Evaluation Research (CDER) and European Medicine Agency (EMEA) for bioanalytical method.

Leech saliva contains biologically active compounds that are mainly proteins and peptides. In Malaysia leeches have been used for traditional medicine for a long time. However, there are scanty studies about the isolation and characterisation of Malaysian leeches` saliva. This study aimed to isolate and characterize leeches saliva extract. A modified and smooth extraction method of leeches` saliva without leeches` scarification is used. UV and Bradford assay protein methods showed that the saliva extract contains high concentrations of protein. RP-HPLC chromatogram revealed that more than 30 different peaks were there in leech saliva extract.. Gel electrophoresis revealed the existence of protein and peptides with different molecular weights. The gel showed up to 25 different bands. Comparison of gel electrophoresis data with protein database revealed the closeness of many molecular weights to known proteins isolated from the Hirudinaria leech family. Other proteins detected by gel electrophoresis may be related to completely new biologically active proteins and peptides or to a modification (isoforms) of the existing ones. It was observed that the period of starvation has a vital role in the concentration of saliva proteins and 12 weeks of starvation gave the highest concentration of proteins in the saliva. It was found also that 4 weeks of starvation after first feeding is enough for leeches to recover 42% of their protein concentration. Two anticoagulant proteins (protein 1 and protein 2) were isolated from leeches saliva extract by using RP-HPLC, and their molecular weights were identified (6.289kDa and 14.255kDa) respectively using tricine SDS-P AGE. These two proteins increased the thrombin time by 29 .11 % and 44.13% respectively. In addition, they inhibited the amidolytic activity of thrombin, evaluated by measuring the conversion of the chromogenic substrate (S-2238). The result showed a decrease in the conversion of the substrate (S-2238) by 30.61% and 41.22 % respectively. Traces of heavy metals concentrations were investigated in the water (natural habitat of the leeches), leeches` tissues and leeches` saliva extracts. The concentrations of heavy metals in the leeches` habitat water were found high. Hence the water specification is a class IV (INWQS). Furthermore, traces concentrations of heavy metals were found in leeches` tissues as well as in their saliva extracts. Such concentrations of heavy metals may cause health hazard, especially when leeches from such contaminated environment are applied in treatment and therapy directly without any precautions. Clearing leeches and their saliva extracts from these traces is investigated in this study. It was found that these high concentrations can be mitigated by successive replacement of the lake water by clean and non-chlorinated or distilled water for three weeks. A significant decrease for certain heavy metals concentrations was achieved depending on the type of metal. For instance in the saliva extract, undetected level of cadmium (Cd) was observed while a marginal 7.3% decrease in the case of arsenic (As) was reported after washing. In the case of leeches` tissues, the concentration of cadmium (Cd) increased unexpectedly, while a decrease of 92.38% and 20.01% in the concentration of lead (Pb) and arsenic (As) were recorded respectively.