Transitioning existing LC methods to new technologies

Analytical laboratories face the continual challenge of balancing investment in new technology – to improve throughput and performance – with the need to run existing methods and tests to support current production. High Performance Liquid Chromatography (HPLC) is one of these key analytical technologies.

• In the pharmaceutical industry, HPLC is employed throughout the whole drug analysis process, including drug discovery screening, raw material analysis, impurity profiling, stability studies, pharmacokinetic studies and final product testing.
• HPLC has found great application in the environmental and chemical analysis arena, where it has been employed for testing water quality, soil samples and product quality.
• Food safety analysis has also benefited from the capabilities of HPLC either alone or in combination with mass spectrometry (MS), allowing food to be rapidly assessed for contaminants such as pesticides or veterinary drugs.

Over the last two years, several instrument manufacturers have introduced LC systems that are designed to take advantage of columns packed with smaller particles. Compared to traditional LCs, these instruments are capable of higher pressure operation, have reduced system volumes, use faster autosamplers and employ detectors with much higher data capture rates.

All of these elements are required to leverage the benefits of higher resolution, sensitivity and throughput offered by columns packed with smaller particles. R&D and method development laboratories have extensively adopted these new LC platforms, as they greatly improve the efficiency of product development.

Methods for products advancing through the pharmaceutical pipeline are now being developed with these new LC platforms. Thus, quality control (QC) laboratories must also make similar technology investments to enable them to run the methods that will be required to release new products. Furthermore, many QC labs are in the process of replacing traditional instruments purchased five to seven years ago that have fully
depreciated.

The convergence of these two trends leaves QC lab managers at a critical decision point with respect to investing in a platform for the future. Assuming a depreciation cycle of five years, the platform chosen today must remain relevant until 2012.

Such a long-term view makes selecting one of the new LC platforms attractive. However, given that the chosen platform will predominantly be running legacy methods during its early lifecycle, it is critical that it can effectively run these methods. At the same time, it must be fully capable of running methods being developed today with small particle columns – as well as methods developed in future years that will utilize next-generation columns. A new LC platform, using Ultra Performance LC (UPLC) Technology, can be used to run these legacy methods.

Illustrating the advantages of Acquity UPLC

The Waters ACQUITY UPLC System, when used in combination with the 1.7 µm stationary phase, increases chromatographic performance. This is illustrated in the analysis of Omeprazole, a proton pump inhibitor (PPI), which blocks the production of acid by the stomach.

Figure 1 illustrates the chromatography obtained on a 4.6 x 150 mm, 5 µm C8 column eluted under isocratic conditions with a phosphate buffer: acetonitrile (3:1) mobile phase at a flow rate of 0.8 mL/min.

The data in Figure 2 illustrates that, when scaled correctly and the same column length is used, the UPLC solution produces superior peak shape and sensitivity. Other major benefits of increased performance are the sharper peaks; they not only increase resolution and sensitivity but also simplify the task of peak integration, reducing the need for manual reintegration.

Anther advantage of using UPLC in the field of product release testing is the ability to shorten analysis time without reducing peak resolution. This is achieved by scaling the separation from the existing LC methodology to UPLC by keeping the ratio of column length to particle size (L/dp) constant. This increases throughput without compromising analytical performance.

Figure 3 shows the separation of the common beta1-selective (cardioselective) adrenoreceptor blocking agent Atenolol, using a 3.9 x 300 mm, 5 µm C18 column. The column was eluted at a flow rate of 0.52 mL/min with a mobile phase of 70 percent phosphate buffer (pH 3.0) and 30 percent methanol. Detection was performed by UV absorbance at 226 nm.

The inset table in Figure 3 illustrates the assay reproducibility of the separation performance. The retention time variation was 0.47 percent RSD and the peak area deviation calculation returned a value of 0.3 percent.

The result of scaling this separation to ACQUITY UPLC technology can be seen in Figure 4. The analysis time has been significantly reduced – from 7.8 minutes to less than 1 minute – while the assay performance remains unchanged in terms of plate count.

Reducing analysis times and increasing chromatographic performance with UPLC can be achieved by reducing the column length by a factor of two while reducing the particle size by a factor of three, from 5 µm to 1.7 µm. The flatter nature of the van Deemter plot for the 1.7 µm material allows a faster mobile phase linear velocity to be employed, further speeding up the analysis.

This approach is illustrated in the analysis of Budesonide, a common inhaled steroid; the chromatogram shown in Figure 5 depicts the separation of the two components on an ACQUITY UPLC System using a 4.6 mm x 250 mm, 5 µm C18 column with a mobile phase of 68 percent 20 mM ammonium formate buffer (pH 3.2) and 32 percent acetonitrile. Detection was performed by UV absorbance at 240 nm.

Figure 5 shows that there are small low-level impurities as well as the R- and S-epimers of the active component. The assay was transferred to an ACQUITY UPLC BEH 2.1 x 100 mm, 1.7 µm C18 column operating with the same mobile phase and a flow rate of 0.6 mL/min. The resulting chromatogram is displayed in Figure 6.

The analysis time has been reduced from 25 minutes to 10 minutes; the chromatographic performance has also improved, producing sharper peaks. The extra chromatographic resolution and increased sensitivity with UPLC has allowed the detection of more impurities in less time, while the resolution of the two major peaks has been maintained.

An excellent platform

UPLC is a new category of separation science that builds upon the well-known and established principles of liquid chromatography. It leverages the performance of sub-2 µm particles to provide increased resolution, sensitivity and throughput. The technology has been specifically designed to exploit the capabilities of these new chromatographic stationary phases, with detailed attention focused on controlling system volumes and peak dispersion. Although this system has been designed to work with these new small particles, it also provides an excellent platform for easily transferring existing HPLC methods to the new UPLC methods. Thus, this new technology is allowing users to confidently move from existing LC methods to the new technology of UPLC.

Waters, ACQUITY UPLC, UltraPerformance LC and UPLC are registered trademarks of Waters Corporation. The Science of What’s Possible is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.