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Design of experiments was used to study robustness of the method. A 24 factorial design was used to test the robustness of chromatographic separation. The experimental design is useful for this kind of study as it facilitates the investigation of several parameters by reducing the number of experiments. Acetonitrile content of the mobile phase, pH, column oven temperature, and R428 cell line flow rate was investigated. Upper and lower limits are shown in Table 5. The experiments were run randomly with sample containing gemcitabine and internal standard (20??g/mL of each). The selected responses were resolution (Rs), tailing factor of drug (Tf-D), and tailing factor of IS (Tf-I). Table 5 Factorial design data for robustness of chromatographic separation. 2.5.5. Stability Studies Stress study like oxidative, alkaline, and acidic stress, exposure to sunlight and UV light (254?nm), was carried out using raw material. Chromatograms JQ1 research buy were recorded in order to study the specificity of the method. The chromatograms of the samples were compared with those of control samples that were freshly prepared from the stock standard solution and without stress. All samples were analyzed in triplicate. The peak purity was checked using the tools of the LC-Solution software. This assessment was based on the comparison of spectra recorded during the elution of the peak. UV spectra and peak purity were used to assess purity of analytes. (1) Oxidative Stress. Gemcitabine (5?mg) was weighed accurately and transferred to 100?mL flask for evaluation of oxidative stress. 10?mL of 5% hydrogen peroxide was added to it. It was shaken for one hour at 60��C and then contents were cooled to room temperature. Internal E-64 standard (2?mL) was added; the contents were transferred quantitatively to 100?mL volumetric flask and diluted to the mark with mobile phase. (2) Effect of Acidic, Alkaline, and Aqueous Media. Similarly for evaluation of acidic, alkaline, or hydrolytic stress, gemcitabine (5?mg) was weighed accurately and transferred to 100?mL flask. These samples were shaken (at 60��C, 1?h) with either hydrochloric acid (5?mL, 1?N HCl) or sodium hydroxide (5?mL, 1?N NaOH or 20?mL water). After one hour the content was cooled and processed as described above (in oxidative stress). (3) Effect of UV Light or Sunlight. Gemcitabine (500?mg) was placed in an open watch glass and exposed to either UV-irradiation (~100?W/m2) or direct sunlight for one hour with occasionally shifting of the content using stainless steel spatula. After exposure, 5?mg of sample was weighed and transferred to 100?mL volumetric flask; internal standard (2?mL) was added to it and further processed as described above. Chromatograms of these sample solutions were recorded and compared with the chromatograms of unexposed API.