HPLC gets faster

Michael G Frank, applications chemist at Agilent Technologies in Germany, explains how the latest high speed chromatography devices can speed up QA/QC

Pre-shipment surveillance of manufactured drugs is a regulatory requirement instituted to protect the consumer. It also serves as an early warning to the manufacturer of problems that may arise with raw materials, intermediates or the manufacturing process. Drug analysis methods are developed to meet exacting standards and must be validated and then carried out with high precision.

When the time to complete all the ancillary procedures is taken into account, an analysis with a modest run time of 30-60 minutes can require more than an entire day for completion. Table 1 lists a typical series of analyses that must be performed to verify that a sample meets the required purity standard.¹

Reducing QA/QC analysis run times would enhance manufacturing productivity and profitability. Accelerating the rate of batch approvals translates into higher use of manufacturing capacity and labour, faster raw material turnover and more timely product distribution. Notwithstanding the benefits, accelerated analysis run times are acceptable only if the method also meets the requirements for chromatographic peak capacity, selectivity and sensitivity. This is now possible using Sub-2-µm particle stationary phases and the requisite instrumentation.

As one moves to smaller particle sizes, chromatographic efficiency increases. Normally the efficiency gains diminish as the flow rate is increased. This is not the case for Sub-2-µm particles, which maintain high efficiency over a broad linear flow range (see figure 1). Therefore, given an instrument capable of generating the required flow, Sub-2-µm chromatography offers the possibility of developing methods with dramatically shorter run times with comparable or better resolution (Figs. 2A & 2B). This is especially important for pharmaceutical QA/QC assays that rely on retention time reproducibility using just two UV detection wavelengths for analyte confirmation.

Sub-2-µm LC operates in a pressure and flow rate regime considerably more demanding than traditional HPLC and requires LC technology with extended performance capabilities. Pumps must be able to generate flows up to 5ml/min and, as a result, the conventional LC system configuration requires considerable re-engineering. At higher flow rates, controls must be more precise and greater attention needs to be directed towards preserving sensitivity. Both the individual system components and the system integration must be sufficiently rugged to withstand the higher stresses without compromising performance or requiring frequent maintenance.^2,3

Fast methods based on Sub-2-µm LC can easily have run times 10-15 fold faster than conventional HPLC methods. Moreover, the gain in run time can often be achieved with additional increases in resolution and sensitivity. The most versatile instrument platforms allow for the manipulation of parameters, such as temperature, in order to further multiply the efficiency gains inherent in the Sub-2-µm stationary phase. Run time reductions as great as 20-30 fold are attainable for Sub-2-µm methods running at temperatures as high as 100°C.⁴

While reducing run time is often the primary objective for using this technology, there may also be circumstances in which achieving higher resolution is the more important requirement. One instrument provision useful in this regard is the design of column compartments with sufficient space to run longer columns or to couple short columns together for the same purpose.

Typically, the QA/QC check must meet international industry guidelines which have been set by regulatory agencies such as the US FDA and the European EMEA. For example, the ICH Q3A guideline of the International Conference for Harmonisation requires that for a drug formulation with a daily dose below 2g/day all impurities at concentrations >0.1% of the active ingredient must be identified and quantified, while impurities with concentrations >0.05% must be reported (but need not be identified). This 0.05% threshold is lowered to 0.03% for drugs taken in daily doses exceeding 2g and the 0.1% identification threshold is lowered to 0.05%.

A validated method meeting these requirements must also meet a host of sensitivity and precision standards. Table 2 shows that the S/N for each of the assayed analytes in figure 3 (top right) exceeds the limit of quantitation (LOQ) cut-off for current pharmaceutical impurity analyses.⁵ Given the trend toward lower LOQs and the fact that newer generations of pharmaceuticals are typically administered at lower dosages, the ability to exceed currently required detection limits is seen as both forward looking and salutary.

reduced noise

High-speed chromatography with good resolution will produce sharp, rapidly eluting peaks. To preserve the quality of the separation, Sub-2-µm LC instruments may incorporate additional noise limiting technology. Examples include pump ripple dampening to reduce mixing noise, low-noise electronics and a noise reducing flow cell design. When running chromatography at elevated temperatures, rapid post-column cooling can be employed to lessen signal noise generated in the flow cell and to reduce the possibility of analyte degradation by shortening the residence time in the hot mobile phase. Sensitivity and selectivity can be improved further by utilizing recently developed high sampling rate diode array detectors. Even at the high data rates typically required for Sub-2-µm LC methods these detectors easily achieve a S/N >10 at 0.5mAU signal height, which translates to a 0.03% impurity limit of detection (LOD).⁴

The capacity for a Sub-2-µm LC instrument to accept and run conventional HPLC methods on standard bore columns ( >3.0mm I.D.) is very important for pharmaceutical applications. While the development of fast QA/QC methods may be desirable for the analysis of newly commercialised products, managers may be reluctant to abandon existing validated methods that do not demonstrate a favourable cost-benefit analysis. Moreover, the work and time involved in method conversion may constitute an unacceptable interruption in the highly regulated workflow. An instrument with the capability of running both standard HPLC and Sub-2-µm LC in standard bore columns addresses these concerns and also facilitates migration from conventional to rapid LC methods with minimal workflow interruption (figs. 2 & 3).4 Currently, the only instrument with this capability is the 1200 series Rapid Resolution Liquid Chromatograph (RRLC) from Agilent Technologies.

Instruments performing regulated analyses must comply with all of the 21 CFR Part 11 regulations such as authenticated signatures, chain of custody documentation, accessible audit trails, privileged access, and system/method validation. In addition to meeting all of these requirements, technology may be deployed to enhance both system and network security. This includes Internet firewalls and data encryption plus strategically located radio frequency tags (RFIDs) that can capture system settings and sample IDs, and protect against data loss in the event of a communications interrupt.

At least one of the new Sub-2-µm LC instrument platforms also incorporates "intelligent functions" to help maximise productivity.² Examples of these functions include system monitoring software that alerts the user when part replacement or a system adjustment is indicated and diagnostic programs that enable the user to quickly resolve problems without requiring a high level of chromatographic experience or the intervention of a service engineer.

Acknowledgements:

Thanks to Professor Frank David for access to fast LC data on drug impurities.