A total of 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50mM phosphate buffer (pH 7). The reaction was started by the addition of 1 ml freshly prepared 30mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm. Catalase values were expressed as n moles H2O2 consumed/min/mg protein. Measurement of lipid peroxidation TBARS, a measure of lipid per oxidation, was measured as described by Ohkawa .
2.2 Chemicals and reagents The API of AN (99.9% pure) 1000mg was purchased from market. HPLC grade acetonitrile (SD fine limited). Analytical grade hydrochloric acid ,sodium hydroxide flakes, hydrogen peroxide. Milli-Q Water purchased from market.. 2.3 Details of Method Chromatographic conditions: Reversed Phase High Performance liquid chromatography method with UV detection separation was achieved on zorbox Agilent Eclipsc XDB column c18(150 nm× 4.6 mm×5µm) as stationary phase with binary gradient mode solvent phase A. Composed of H3PO4(ortho phosphoric acid ) buffer ( pH ≈2, 0.02M) and phase B as Acetonitrile ,The Flow rate of the mobile phase was 1.0 mL/min and the total elution time including the column re-equilibration was approximately
Twenty tablets were weighed accurately and powdered. An amount of the powder equivalent to 5 mg of amoxicillin trihydrate (content of one tablet) was dissolved in 60 ml of diluent. The solution was stirred for 10 min using a magnetic stirrer and filtered into a 100 ml volumetric flask through 0.45µ nylon membrane filter. The residue was washed 3 times with 10 ml of diluent and then the volume was completed to 100 ml with the same solvent. This solution was diluted with diluents to gae a concentration of 0.1 mg/ml solution each of Amoxicillin trihydrate.
Upon finding the actual concentrations of salicylic acid, concentration of aspirin in the flask at various times can be found using the equation [aspirin]t = [aspirin]0 – [salicylic acid], since at constant volume, number of moles of initial aspirin decrease to form salicylic acid. Initial concentration of aspirin formed as follows: [aspirin]0 = 0.212g / (180.157gmol-1 * 50/1000 L) = 0.0235 mol L-1. Thus using the first test as sample, [aspirin]t = 0.0235 – 9.981*10-4 = 0.0225 mol L-1. To find the rate constant, we will need to log the value of [aspirin]t and plot it against time to find the rate constant. Figure 1 shows the diluted and actual concentrations of salicylic acid, the concentration and log value of aspirin at various times.
Standardization of NaOH solution Accurately weigh out a sample of approximately 0.3-0.4 g of primary standard potassium hydrogen phthalate, KHPh, which has been previously dried at 120°C. Do not use more than 0.4 g. To obtain an accurate mass, weigh the sample on weighing paper, slide it into a clean (but not necessarily dry) 250 mL Erlenmeyer flask and reweigh the paper to account for any KHPh that may remain on it. Dissolve the KHPh sample in about 50 mL of CO2-free water and add 2-3 drops of 0.1% phenolphthalein indicator. Begin adding the approximately 0.1 M sodium hydroxide solution from the buret while continuously swirling the flask contents. Do not open the stopcock completely.
Arrehnius did not explain how acids and bases behave in a non aqueous solution. For example, there is dissociation of acetic acid in methanol: CH3CO2H + CH3OH ⇄ CH3CO2− + CH3OH 2. He claimed that an acid should be an acid in any solvent. However, there is rebuttal nowadays. For example, dissolving in water, HCL behaves as Arrhenius acid but dissolving in benzene there is no dissociation.
S. DRUG SUBSTANCE S1. General Information S1.1 Nomenclature Table 84 Nomenclature of Drug Substance International Nonproprietary Name (INN) Levofloxacin Hemihydrate Chemical Name 7H-Pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-hydrate (2:1), (S)-. (-)-(S)-9-Fluoro-2,3-dihydro-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7Hpyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, hemihydrates Generic name Levofloxacin Hemihydrate CAS Number [138199-71-0] S1.2 Structure and attachment for structure of drug substance Table 85 Structure of Drug Substance Structural Formula : C18H20FN3O4 •½ H2O Relative Molecular Mass : 370.38 Structural Formula : S1.3
Filtered the solution through 0.45 µm nylon filter Buffer having pH 3.70 used as Mobile phase A and mixture of methanol, acetonitrile and tetrahydrofuran (50: 50: 2 v/v/v) were used as Mobile phase B. Diluent: Prepared a mixture of buffer and methanol (80: 20 v/v). Placebo preparation (Placebo I): Weighed accurately and transferred 255 mg of placebo to a 100 mL volumetric flask. Add 40 mL of methanol and sonicated for 10 mins and add about 30 mL of diluent and again sonicate for 15 mins. Allow to equilibrate at room temperature (RT) and dilute to volume with diluent. Filter the solution through 0.45 µm nylon filter (25 mm) by discarding first few mL of the filtrate.
1 ml = 1 µg CN (x) Chloramine –T: Dissolve 1 gm chloramine – T in 100 ml distilled water. Prepare fresh solution daily. (xi) Pyridine (xii) 1-phenyl–3-methyl– 5-pyrazolone solution: Prepare a saturated aqueous solution (approximately 0.5 gm / 100 ml) by adding the pyrazolone to water at 75 0 C. Agitate occasionally as the solution cools to room temperature. (xiii) Bis–Pyrazolone (3,3-dimethyl-1-diphenyl) (4,4’-bis-2-pyrazolone)-(5,5’
Linoleic acid peroxidation was initiated by the addition of 4 mM FeSO4.7H2O, incubated for 60 min at 37oC and terminated by the addition of 2 mL of ice cold trichloroacetic acid (10% v/v). An amount of 1 mL of thiobarbituric acid (1% w/v in 50 mM NaOH) was added to 1 mL of the reaction mixture, followed by heating at 95oC for 60 min. The reaction sample was read at 532 nm.7 The percentage of linoleic acid peroxidation inhibition activity was calculated using the following equation: % Inhibition = [(AB - AA)/AB] x 100, where AB, absorption of blank sample, AA, absorption of test sample. 2.5.4. Metal chelating activity Briefly, 2 mM FeCl2 was added to different concentrations of test sample and reaction was initiated by the addition of 5 mM ferrozine.