Improving the Analysis of Betamethasone and Phosphate, Acetate, and Dipropionate Derivatives Using CORTECS™ Premier C₈ Columns

Newer column technologies can significantly improve analytical performance. This application note demonstrates the improved analysis of betamethasone and its phosphate, acetate, and dipropionate derivatives using CORTECS Premier C₈ Columns with MaxPeak™ High-Performance Surfaces (HPS) Technology. The inert hardware eliminates interactions with metal surfaces, enhancing peak area, shape, and symmetry, particularly for betamethasone phosphate, while maintaining excellent results for the other analytes.

Benefits

• Resolution of four steroid compounds under generic screening gradient conditions
• Narrower, more symmetrical peak and higher peak area for betamethasone phosphate obtained using an inert hardware CORTECS Premier C₈ Column compared to a CORTECS C₈ Column and a solid-core C₈ column from a different supplier

While older column technologies may give adequate results for some analyses, using newer technology can provide better separations and future-proof methods to reduce the risk of time-consuming revalidation. As such, it is important to consider newer column technology like MaxPeak Premier Columns during method development, prior to validation. MaxPeak Premier Columns employ inert HPS hardware technology. The hardware mitigates interactions between analytes and the metal surfaces present in the column.^1–3 This technology is also available in liquid chromatography (LC) systems. Using both a system and a column with MaxPeak Premier Technology provides the most complete mitigation of these interactions.⁴ The analytes most affected by metal surfaces in the column and system are generally those with acidic groups or other moieties than can bind metal ions. When using a mobile phase with a pH of less than about seven, the oxide layer on the surface of stainless steel has a positive charge. This acts as an anion exchange site that can interact with any negatively charged analytes like phosphates, sulfates, and some carboxylic acids.⁵

To demonstrate how MaxPeak Premier Columns can improve a separation, four steroid compounds were selected: betamethasone, betamethasone phosphate, betamethasone acetate, and betamethasone dipropionate. Separations were carried out using three different columns: a CORTECS Premier C₈ Column, a CORTECS C₈ Column, and a solid-core C₈ column from a different supplier. Peak areas, United States Pharmacopeia (USP) tailing factors, and peak width at 4.4% were monitored for all analytes. Comparisons were made across the three columns to assess how the inert hardware impacted the separation.

Sample Description: Stock solutions were created at 1 mg/mL in 1:1 (v:v) water:acetonitrile. A sample mixture containing 50 µg/mL of each analyte was created using water as the sample diluent. The final solvent composition was 12.5% acetonitrile in water.

Steroid phosphates, particularly betamethasone phosphate, have been studied previously and were shown to benefit from inert hardware, such as that used in MaxPeak Premier Columns.⁶ However, no such analysis has been performed using CORTECS Columns, which employ solid-core silica particles that give higher column efficiencies.⁷ To that end, four compounds were analyzed using a CORTECS Premier C₈ Column, a CORTECS C₈ Column, and a solid-core C₈ column from a different supplier. In addition to betamethasone phosphate, betamethasone and two other betamethasone derivatives were included. The structures of the analytes are shown in Figure 1. The compounds all contain the same betamethasone backbone, with alterations to position 21 or positions 17 and 21 in the case of betamethasone dipropionate.

Figure 2 shows the results obtained using the three columns. Betamethasone phosphate showed a narrower and more symmetrical peak with a higher area when using the MaxPeak Premier Column compared to the stainless steel columns. No other major changes in the separation were detected between the columns, which is confirmed by examining the peak areas, USP tailing factors, and peak width values for the analytes on both columns, as shown in Tables 1–3.

The numerical data confirm that only betamethasone phosphate was affected using MaxPeak Premier Column hardware. The best peak shape for betamethasone phosphate was obtained using the CORTECS Premier C₈ Column, with slightly worse peak shape on the CORTECS C₈ Column, followed by the Vendor A C₈ column. The other compounds, which are not susceptible to metal interactions, gave comparable results using the three inert columns.

MaxPeak Premier Columns employ inert HPS hardware to mitigate the interaction between analytes and metal surfaces in the column. These interactions can lead to peak tailing and loss of peak area in a chromatographic analysis. By standardizing on MaxPeak Premier Columns for all new method development activities, any possible interactions between analytes and column hardware will be mitigated, elevating the quality of the data and future-proofing methods.

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