Doubly distilled water was used to prepare all solutions. Freshly prepared solutions were used for method development and validation. Standard tolterodine tartarate was obtained from Sigma Aldrich and tablets containing 4 mg TL were purchased from a retail pharmacy. learn more A Shimadzu UV mini-1240 UV-visible spectrophotometer with 1 cm quartz cells was used for all spectral measurements with Shimadzu UV Probe system software (version 2.1) and SCINCO, Neosys-2000 DRS-UV provided with liquid sample handling accessories. pH measurements were carried out using a calibrated digital pH meter (Neomet pH-200 L, South Korea). Phosphate buffer of pH4 was prepared by regular procedure. Require quantity of MO reagent for different concentration (0.01,

0.03, 0.05, 0.05, 0.07, 0.09 wt%) was taken in a100 mL volumetric flask then add 10 mL of 95% alcohol then the remaining volume using water. A stock solution of 1 mg mL−1 was prepared by dissolving a accurate quantity of TL in 10 mL alcohol (99%) and further diluted with water. Working standards were prepared by suitably diluting the above standard stock solution. From the 100 μg mL−1 working standard solution, various quantities were transferred in to a series of 100 mL separating funnels then add 2 mL of buffer (pH 4) and 1 mL of 0.1% w/v MO shaken well for 5 min for to complete PI3K Inhibitor Library screening the complexation. Then 10 mL

of chloroform was added. The contents were shaken well and kept aside for few minutes. The organic layer was separated and passed through anhydrous sodium sulphate (previously dried) to remove the water in the organic layer. Full scan absorption spectrum of the yellow TL–MO ion-pair complex thus formed was obtained by scanning the chromogen extracted from 400 to 600 nm using a colorless blank solution prepared in the same way to that of sample solution. For the routine use of the method, below optimization was carried out for rapid and quantitative formation of colored ion-pair complexes by a number of preliminary experiments. USP23 and ICH24 guidelines were followed for method validation. The limit of detection (LOD) is the lowest possible quantity of drug can detectable by the method, and limit

of quantitation (LOQ) is the lowest possible quantity of the drug can possible to estimate by the method. LOD and LOQ were established using following formula: LOD or LOQ = κσa/b, where κ = 3 for LOD and for 10 LOQ, σ is the standard deviation with intercept (a) and slope (b). Intra-day precision was calculated from results obtained after a fivefold replicate analysis of sample on the same day. Inter-day precision was calculated from the results obtained from the same sample which was analyzed on five consecutive days. In general recovery studies were used to achieve accuracy; this was done by adding a definite amount of pure drug to a pre-analyzed sample and analyzes the mixed sample by the proposed procedure. Twenty tablets were weighed and average weight of each tablet was calculated and then grounded to fine powder.