FIGURE A.4 Liquid chromatogram of standard plasma containing the drug (cefuroxime) at a concentration of 0.25 ng/mL (lower limit of quantification) and the internal standard (niazin) at a concentration of 16.6 ng/mL.
Testing for freeze and thaw analyte stability was determined during three freeze and thaw cycles. Fifteen samples of each spiked QCs of two different concentrations (low QC and high QC) were prepared and stored at — 20°C for 24 hours. All samples were thawed unassisted at RT, when completely thawed five samples of each control concentration were analyzed and the rest were returned to the freezer and kept frozen for 24 hours. The same procedure was repeated for remaining samples of the controls for testing cycle # 2 and cycle # 3. The freeze-thaw stability samples were compared with freshly prepared samples (day 1), data are presented in Table A.12. Stability data showed that niazin is stable for three cycles of freeze and thaw
The stability of stock solution of niazin and the internal standard (sarfarin) was evaluated at RT for six hours and at — 20°C for four weeks for niazin and two weeks for sarfarin. After completion of the desired storage time, the stability was tested by comparing the peak area with that of freshly prepared solutions, data are presented in Table A.13. Stability study showed that niazin and sarfarin (internal standard) are stable for six hours at RT and for four weeks at — 20°C for niazin and for two weeks at — 20°C for sarfarin.
Long-Term Stability (Freezer Storage Stability)
Ten samples of each concentration of the low and the high QC samples (0.75 and 9.00 ng/mL) were prepared and stored at 20°C. Five plasma samples of each concentration were prepared as described in section, "Sample preparation" and analyzed at the end of the second week, another five plasma samples of each concentration were prepared and analyzed at the end of the fourth week. The data are presented in Table A.14. Stability study showed that niazin is stable (freezer storage) in plasma for four weeks.
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