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The proposed methods are accompanied by the reaction of amine group of amino acid with hydroxyl group, activated by neighboring nitro groups of both TNP and DNP. As a result, yellow color complexes are formed on simple addition of the two reagents and no extra process is required. Thermodynamics features in respect of association constant and standard free energy changes have been evaluated for both reactions. Both of methods have been proved to be very useful from routine quality control prospective and can be considered as superior to most of the published methods with JQ1 in vivo respect to pace, ease, low cost, and sensitivity. 2. Experimental 2.1. Instrument A UV-Visible Shimadzu Spectrophotometer 1601 with 1?cm path length quartz cells controlled by Shimadzu UV Probe 3.9 version software was used. MS Excel sheet was used to E-64 evaluate F, t tests, and other statistical parameters. 2.2. Materials and Reagents Analytical grade reagents were used. Pregabalin and tranexamic acid pure drugs were a kind gift from a local pharmaceutical agency having 99% plus purity. Tranex capsules 250 and 500?mg and Syngab capsules 200?mg, 50?mg, and 100?mg (Atco Laboratories Ltd., Karachi, Pakistan) were procured from the market. TNP and DNP were acquired from Merck Sigma Aldrich, Germany. Microcrystalline cellulose (Avicel pH 101, maize starch, magnesium stearate, PVP, Aerosil R-200, crospovidone-X, HPMC (606), and PEG (6000)), titanium dioxide, and isopropyl alcohol were a gift from a local pharmaceutical agency. The entire chemicals were used according to their safety precautionary measures. 2.3. General Procedure 2.3.1. Analytical Method Development Reagents and Standard Stock Solutions Preparation. A 2.27?gL?1 TNP and 1.84?gL?1 2,4-DNP solutions were prepared individually in dichloromethane corresponding selleck chemicals llc to 10?mMol of the concerned reagent. 160?��gmL?1 of PG and 155?��gmL?1 of TXA were prepared individually by taking 16 and 15.5?mg weights, respectively, in 100?mL calibrated volumetric flasks, dissolved in 10?mL of water, and diluted to the mark level with the same solvent. Stoichiometric Study. To study stoichiometry of the reaction, Job's method of continuous variation was followed [31]. Equimolar solution (1?mMol) of each of the coloring reagents and drugs were prepared. In a series of 10?mL calibrated amber volumetric flasks, solutions of PG and TXA were prepared individually, comprising different complementary proportions (0?:?10, 1?:?9, 2?:?8, 3?:?7, 4?:?6, 5?:?5,��, 9?:?1) each with both of the reagents (TNP and DNP) separately. After 10 minutes of reaction the absorbance of the complex was measured at the wavelength of maximum absorption against the reagent blanks treated similarly. DNP Method. 3?mL of drug PG or TXA stock solution was taken individually in 10?mL amber volumetric flasks to which 4?mL of the coloring reagent DNP was added.
