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    The linear regression equation was used to calculate the concentrations of GFX in spiked urine based on their peak area ratios. Method Validation The proposed analytical method was fully validated in terms of sensitivity, linearity, stability, selectivity, accuracy, intraday and interday precision and system suitability.

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    The method validation was developed based on the criteria established by the recommendations of the International Conference on Harmonisation ICH. Specificity Specificity is the ability of a method to determine accurately the analyte of interest in the presence of other components in a sample matrix under the stated conditions of the test.


    To demonstrate the specificity of the analytical procedure, ten different blank urine samples obtained from various healthy volunteers were analyzed for the peaks interfering with the detection of the analytes or internal standard.

    Possible carryover effects were reduced via the use of MRM mode and increasing run time after elution of the analytes.

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    Linearity and Sensitivity Using the aforementioned optimum chromatographic conditions, three independent calibration curves were constructed correlating the calculated peak area ratio of GFX to the internal standard OFX versus the nominal concentrations of GFX.

    Calibration plots for GFX in urine samples were prepared daily at eight concentration points; each concentration was injected in triplicate. Regression analysis for the results was carried out using the least-square method see Section 2.

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