Despite remarkable improvements in the speed of high performance liquid chromatography (HPLC) over the last decade, due to the development of columns containing sub-2 μm particles and ultra-high performance liquid chromatography (UHPLC) instrumentation, chromatographic method development remains a significant bottleneck in many analytical laboratory workflows. Developing suitable chromatographic conditions can take weeks or even months, especially if extensive column and eluent optimization are required. This can limit laboratory productivity and increase operational budgets through the extensive use of costly resources such as solvents, even when systematic approaches are employed.

The latest automated method scouting techniques offer a solution to these problems, and can be used to truly leverage the ultra-fast analytical speeds of UHPLC. These systems combine automated multi-column and solvent screening and intelligent run analysis software, which allow chromatographers to develop effective (U) HPLC methods more rapidly, saving valuable time and resources. In this article, we look at how the technology can be used to develop a suitable UHPLC method for the separation of two isomers of budesonide, a steroid used in the long-term treatment of asthma.

Multi-parameter optimization

When developing a new reversed-phase HPLC method, several parameters must be considered. In addition to the selection of an appropriate column—which can include factors such as stationary phase chemistry, particle size and column length— other important conditions, including mobile phase pH, separation temperature, solvent choice and gradient profile must also be systematically optimized.

Eluent pH is a particularly important factor to consider when analyzing ionizable compounds to ensure these analytes remain in their desired ionization state. In such cases, the mobile phase pH should be adjusted according to the pKa value of the compound, which can be challenging for mixtures of acidic and basic compounds and often necessitates screening buffered eluents of varying pH.

Appropriate column selection is also very important for reversed-phase HPLC. For polar compounds, the selectivity may vary between different C18 column chemistries depending on endcapping and additional polar selectivities. Endcapping is used to remove surface silanol groups on the solid support that can cause undesirable secondary interactions between the analyte and column resulting in poor peak shapes, particularly for ionizable and charged analytes. It can therefore be helpful to investigate separation on a range of C18 columns.

An additional and often overlooked parameter that is worth considering during method development workflows is the separation temperature. This can change the selectivity dramatically. Resolution of mixtures of enantiomers and labile compounds, for instance, can benefit from separation at sub-ambient temperatures.

If all of these parameters are to be considered and optimized during method development, a large number of chromatographic runs are typically required. Method scouting using an instrument and software package that can automate and analyze chromatographic runs can therefore simplify method development considerably. To demonstrate the power of this approach, a chromatographic method for the separation of therapeutic steroid budesonide was developed.

Figure 1. Instrument flow scheme.

Instrument set-up

A Vanquish Horizon UHPLC system (Thermo Scientific) in combination with Chromeleon 7.2 Chromatographic Data System software were employed for method development. The UHPLC system was fitted with an optional solvent reservoir extension kit, which offered the potential for binary solvent blending with multiple solvent options. The solvent reservoir extension kit utilizes a 10-port valve in line with one of the three available solvent lines from the pump to add an additional nine buffer options. This permitted up to 12 aqueous mobile phases and three organic mobile phases to be used, allowing users to easily switch between mobile phases for rapid method development. Additionally, the system possessed two six-position, seven-port switching valves that allowed up to six columns to be considered. The instrument flow scheme is shown in Figure 1.

To develop a suitable separation protocol for the two epimeric forms of budesonide (0.5 mg/mL in 1:1 acetonitrile–water), four different reversed-phase columns of varying chemistries were scouted with six different aqueous buffers from pH 3 to pH 8. While the Vanquish Horizon system permits up to three different organic solvents to be used, in these experiments, only acetonitrile was utilized as the organic solvent. Using passive pre-heaters, the separation temperature was maintained at 30 °C for all four columns. Full experimental conditions are shown in Table 1.

Table 1: Experimental conditions. Source: Thermo Fisher Scientific.

Method scouting workflow and data evaluation

An eWorkflow was downloaded from the Thermo Scientific AppsLab Library of Analytical Applications1and was customized in the Chromeleon software to accommodate parameters used in this study, such as active column position, aqueous buffer, flow rate and solvent selection. All of the sequence setup parameters could be modified in a single, convenient, method development interface.

The intelligent run control features in the software were used to check which injections passed certain pre-defined criteria, which included the number of detected peaks, minimum resolution between critical peaks, and peak asymmetry. Separation data from runs which passed these criteria were visualized using the software’s query tool, which could be used to plot retention time and peak resolution as a function of the chromatographic parameters. In total, 24 different chromatographic conditions were tested within 20 hours, and required no manual interaction.

Effect of run conditions on separation performance

The retention times for the two epimers on all four columns were found to be similar, and were unaffected by the pH of the eluent. Representative chromatograms using the four columns tested are shown in Figure 2. The Accucore Vanquish C18+ column, containing 1.9 μm solid core particles, gave the best resolution, and was the only column that produced a resolution of more than 1.5, as required by the United States Pharmacopeial Convention for budesonide. This column also offered an enhanced signal-to-noise ratio 20 percent greater than the next best separation method, due to the decreased peak width achieved using the column.

The ability of the Accucore Vanquish C18+ column to separate the two budesonide epimers was further optimized by investigating the effect of separation temperature on peak resolution. With its passive eluent pre-heater that eliminates thermal mismatch, the instrument permitted a range of temperatures to be studied. At sub-ambient separation temperatures, further improvements in resolution could be achieved. Whilst only a marginal improvement in separation performance was observed between 10 and 20 °C, the highest resolution of 2.2 was obtained at an eluent temperature of 10 °C.

Figure 2: Comparison of the separation of budesonide epimers with four different columns during method development. Source: Thermo Fisher Scientific.


(U)HPLC method development presents a significant bottleneck in analytical laboratories, as multiple parameters, including column chemistry, eluent pH and separation temperature often need to be systematically investigated and optimized. We have demonstrated an automated and rapid UHPLC method scouting procedure for the separation of two therapeutic steroid isomers using the Vanquish Horizon UHPLC platform, which allows up to six columns and an extensive range of solvents to be screened for suitability. Additionally, we demonstrated that using a comprehensive software package, system set-up and run analysis could simplify method development. This automated method scouting approach can save time and resources, resulting in increased productivity compared to conventional, manual approaches.