FDA Perspectives: Scientific Considerations of Forced Degradation Studies in ANDA Submissions. (Part-03)
| Analysis method The preferred method of analysis for a stability indicating assay is reverse-phase high-performance liquid chromatography (HPLC). Reverse-phase HPLC is preferred for several reasons, such as its compatibility with aqueous and organic solutions, high precision, sensitivity, and ability to detect polar compounds. As part of method development, a gradient elution method with varying mobile phase composition may be carried out to capture early eluting highly polar compounds and highly retained nonpolar compounds. Stressed samples can also be screened with the gradient method to assess potential elution pattern. Sample solvent and mobile phase should be selected to afford compatibility with the drug substance, potential impurities, and degradants. Chromatographic profiles of stressed samples should be compared to those of relevant blanks (containing no active) and unstressed samples to determine the origin of peaks. The blank peaks should be excluded from calculations. The amount of impurities (known and unknown) obtained under each stress condition should be provided along with the chromatograms (full scale and expanded scale showing all the peaks) of blanks, unstressed, and stressed samples. Additionally, chiral drugs should be analyzed with chiral methods to establish stereochemical purity and stability. The analytical method of choice should be sensitive enough to detect impurities at low levels (i.e., 0.05% of the analyte of interest or lower), and the peak responses should fall within the range of detector’s linearity. The analytical method should be capable of capturing all the impurities formed during a formal stability study at or below ICH threshold limits. It should be noted that structural characterization of degradation products is necessary for those impurities that are formed during formal shelf-life stability studies and are above the qualification threshold limit. Various detection types can be used to analyze stressed samples such as UV and mass spectroscopy. The detector should contain 3D data capabilities such as diode array detectors or mass spectrometers to be able to detect spectral non-homogeneity Peak purity or peak homogeneity of the peaks of interest of unstressed and stressed samples should be established using spectral information from a diode array detector. Limitations to peak purity arise when co-eluting peaks are spectrally similar, or below the detection limit, or a peak has no chromophore, or when they are not resolved at all. Mass balance establishes adequacy of a stability indicating method though it is not achievable in all circumstances. Some attempt should be made to establish a mass balance for all stressed samples. Mass imbalance should be explored and an explanation should be provided. Varying responses of analyte and impurity peaks due to differences in UV absorption should also be examined by the use of external standards. Potential loss of volatile impurities, formation of non-UV absorbing compounds, formation of early eluants, and potential retention of compounds in the column should be explored. Alternate detection techniques such as RI LC/MS may be employed to account for non-UV absorbing degradants. |