(T1530-06-37) A Study of the Effects of Water and other Modifier Additives on the SFC Separation of Ritonavir

Purpose: The use of SFC as a separation technique for achiral molecules continues to expand. With advantages in the realm of purification and alternate selectivity, SFC is being used increasingly in the preclinical space for applications related to Medicinal Chemistry, ADME/DMPK, and Bioanalysis. In this study, we examine various separation conditions in the SFC mode for Ritonavir, an HIV-protease inhibitor therapeutic.

Methods: To examine the effects of polar additives, particularly water, to the modifier, isocratic separations were evaluated using the Shimadzu Nexera UC Analytical SFC system. For all comparisons presented, the isocratic conditions were 80:20, CO₂:modifier. Three modifier mobile phases were made for screening, namely: Methanol (MeOH) with 10 mM ammonium formate (AmFor) [A]; 90% MeOH + 10% H₂O with 10 mM AmFor [B]; 90% MeOH + 10% H₂O with 10 mM AmFor + 0.3% ammonium hydroxide (AmOH) [C]. Using the Solvent Blending function, modifier [A] was blended against [B] and [C] to produce varying concentrations of water, from 1% to 5%. For example, to obtain an effective at-column solvent composition of 95% MeOH + 5% H₂O with 10 mM AmFor, the specified composition for Solvent Blending is set to 50% [A]:50% [B]. This has the effect of diluting the AmOH additive, but allows for rapid exploration of a wide range of mobile phase conditions. Three stationary phases were also screened, namely: Diol (150 mm x 2.1 mm, 3 micron); Pi-naphthyl (250 mm x 4.6 mm, 5 micron); Amino (250 mm x 4.6 mm, 5 micron). Total flow rate was 2.0 mL/min for all experiments, back pressure was regulated at 150 bar, and column oven temperature was set to 50 C. Detection was by photodiode array, with representative 2D chromatograms at 254 nm. Ritonavir was prepared at a concentration of ~1 mg/mL in modifier [A] from dry standard.

Results: Both the Amino and Pi-naphthyl phases exhibited predictable increases in retention of Ritonavir as the proportion of water in the modifier was increased at the collected data points, 0, 1, 3, and 5% of modifier (0, 0.2, 0.6, and 1% of total mobile phase, respectively). While the Diol phase was unpredictable, demonstrating almost no change in API peak top retention from 0-1% water, a shift to earlier elution at 3% water, and splitting of the peak with later elution at 5% water. Examining the resolution of the API to a related impurity detected by PDA, the addition of water was seen to increase resolution on both the Amino and Pi-naphthyl phases, with the Amino column providing earlier elution of the API with better peak shape and increased resolution to the related impurity. The Diol column produced similar resolution to the related impurity regardless of water concentration. Using the Amino column, the addition of ammonium hydroxide along with 5% water, was observed to further increase retention of the API. Resolution to the related impurity was also seen to increase from 12.788 for 5% water without additive to 15.033 for 5% water with 0.15% ammonium hydroxide. It was found that using either modifier [B] or [C] at 100%, effectively 10% water in the modifier or 2% of total mobile phase concentration when running at 80:20 CO₂:modifier, led to a breakdown of homogeneity of the mobile phase preventing detection by PDA.

Conclusion: As SFC separations become more prevalent in Bioanalysis settings, the use of water and other polar additives should be explored to achieve the best separation. When paired with the correct stationary phase, small additions of water and ammonium hydroxide can allow manipulation of retention for representative drugs like Ritonavir to achieve on-column separation from potential interferences. Demonstrating this ability to control retention and resolution character with MS-friendly modifier additives also allows separations developed for SFC to be migrated to SFC-MS.

The effect of water as a proportion of modifier in the SFC separation of Ritonavir using an Amino column. Water concentration expressed as a proportion of the modifier, isocratic conditions 80:20 CO2:modifier.

The effect of ammonium hydroxide to further increase retention of Ritonavir.

The effect of water as a proportion of modifier in the SFC separation of Ritonavir using a Diol column. Note the lack of a trend related to water concentration.