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The stability of stock solution was evaluated at zero time and stored in the refrigerator (2�C8��C). Samples were prepared and analyzed at days 0, 7, 14, and 21. 3. Results and Discussion 3.1. Analytical Method Development In order to achieve optimum separation various parameters like solvent, solvent strength, detection wavelength, flow rate, elution time, asymmetry, and plate numbers were considered. During optimization gemcitabine hydrochloride and internal standard were injected into various mobile JQ1 phases of water?:?methanol or water?:?acetonitrile (90?:?10, 80?:?20, 70?:?30, 60?:??40, and 50?:?50, pH 5, 6, or 7) and the retention time, tailing factor along with resolution factor, was recorded. In certain mobile phases the peak was distorted while in others the compound eluted out quickly indicating the lesser retention time and thus lesser separation on the column. As the pKa of gemcitabine is 3.5 and unstable in an acidic pH [3], different mobile R428 in vivo phases of pH 7.0 were used. Mobile phase of acetonitrile-water (10?:?90, v/v, pH adjusted to 7.0) was used as suitable mobile phase, as it was able to separate the analytes. Using the C-18 ODS analytical column with an isocratic mobile phase at a flow rate of 1 mL/min, the drug and IS were eluted at ~4.0 and 7.8?min, respectively. Although temperature was found not to be a critical parameter for this analysis, it was set at 25��C to avoid shifting of signals. The absorption maximum of the drug at 275?nm was selected for detection (Figure 2), as there was no interference from excipients present in drug or baseline disturbance. The resolution factor was ~11. Figure 3 depicts the representative chromatogram obtained with the present method. Figure 2 UV spectra of the gemcitabine in mobile phase. Figure 3 Representative chromatogram showing signals of gemcitabine and theophylline in the selected mobile phase. 3.2. Method Validation The method was validated with respect to parameters including linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, specificity, robustness, system suitability, and stability. 3.2.1. Linearity Different calibration curves (n = 6) that were constructed E-64 for gemcitabine were linear over the concentration range of 0.5�C50??g/mL. Peak area ratios of gemcitabine to IS were plotted versus gemcitabine concentration and linear regression was performed using Spinchrome-Clarity or LC-solution software. Different calibration curves (n = 6) were prepared on three different days. The mean regression equation for gemcitabine was found to be y = 0.0353x + 0.0063 with 0.9998 correlation coefficient, using weighting factor-x (Table 1). The linearity range reported in other methods ranged between 0.020 and 300??g/mL [3, 5�C28]. Table 1 Linearity data of the proposed method. 3.2.2. LOD and LOQ The LOD and LOQ values were 0.1498 and 0.4541??g/mL calculated using calibration curve as per ICH guideline. The LOD and LOQ reported by Lanz et al.