(T1230-09-58) Systematic Method Development of Deferoxamine Mesylate Forced Degradation Sample

Purpose: Method development activities can be challenging, even with relatively simple mixtures. This is due to the wide variety of available stationary phases, and possible testing conditions. Performing method development without a set plan can lead to lengthy studies without a guarantee of an acceptable method. However, employing a structured method development approach can streamline the process by setting specific rules and guidelines for even novice analysts to follow. This poster will document a systematic method development process for the analysis of deferoxamine mesylate after forced degradation under both acidic and basic conditions.

Methods: Chromatographic methods are run on an LC that is equipped with a quaternary solvent manager and a single quadrupole mass detector, using 2.1 x 50mm analytical columns and mobile phase composition of either acetonitrile or methanol, and either 0.1% Formic Acid or 10mM Ammonium Hydroxide to study the effect of mobile phase composition on the analysis of deferoxamine mesylate after forced degradation. Sample and separation criteria is defined to ensure clear end goals for the method development. In this work the final method needs to fully separate all degradant peaks with USP Resolution values >1.5, and all peaks need to be symmetrical with USP Tailing values between 0.8 and 1.2.

Results: The first step of the systematic screening protocol is rapid scouting, where high and low pH are assessed for overall retention, and from this, we can determine which pH provides the best retention for the probes. For this sample, low pH provided the best retention, with deferoxamine (1) being retained more at low pH. Mesylate (2) is poorly retained under both conditions as it is small polar acid. Next we choose a diverse panel of stationary phases, and we run the chromatographic method using both acetonitrile and methanol mobile phases. The use of acetonitrile mobile phases only provided one acceptable separation, and this separation improved with the use of methanol mobile phases. There was more separation between critical pairs, while still maintaining the peak shape of the analytes. We selected a PFP stationary phase and methanol mobile phase for further study as it gave us the best separation of our analytes.

Conclusion: By implementing a systematic method development process a suitable method was found within two days of analytical runs. The best conditions obtained from method development were subsequently optimized to further improve the cycle time of the analysis. The final method conditions utilized formic acid modified acetonitrile and water on a pentafluoro phenyl (PFP) stationary phase. Employing a systematic method development approach can not only reduce the time needed to develop a suitable method but improves traceability of the method development process and reduces the needed decision making that can accompany method development.

Figure 5. Column screening using methanol mobile phases. EICs of full scan MS used to track peaks

Figure 1. Overview of the systematic screening protocol

Figure 2. Rapid scouting of deferoxamine mesylate forced degradation sample using an XBridge Premier BEH C18 Column. EICs of full scan MS used to track peaks