How to be pure

An effective method for preparing weighable samples from difficult to dry HPLC purification fractions

Over the last 5 years there has been a significant increase in the use of HPLC purification for discovery chemistry samples. Today the technique is probably the purification method of choice within the Pharmaceutical industry. Concurrent with the increasing use of HPLC purification average sample sizes have also increased, meaning that even using mass directed detection more than one fraction is generated for each sample.

Following purification sample fractions are normally dried, re-suspended in minimal solvent and the various fractions pooled into a (small) sample container for storage. Analytical methods are often included in this process, such as dry weight determination of yield and sub sampling for NMR analysis. Until recently, scientists at KuDOS Pharmaceuticals had been experiencing difficulties with samples that did not dry well after purification. The challenges and the solutions to this problem, developed in tandem with colleagues at AstraZeneca, are presented below.

KuDOS Pharmaceuticals, like many pharmaceutical companies, regularly encounter a significant number of samples that do not dry well post purification. Typically, these stubborn samples form a gum or oil (Figure 1) which creates handling problems including:

• An accurate weight cannot be determined - the samples are clearly not fully dry. Consequently the oils and gums are deemed un-weighable.

• Residual solvent promotes faster compound degradation during storage than would be the case with truly dry samples.

A new sample drying methodology, developed by KuDOS/AstraZeneca, that overcomes both the residual solvent removal and fraction pooling problems is detailed in Figure 2.

Using this new methodology the resultant samples are friable "fluffy" solids that can be easily handled and are fully dried. To date this new methodology has been found to work for well over 90% of previously intractable samples. Alternative methods are required for the few samples that do not dissolve well in 1,4-dioxane. Two methods have been successfully used: Either; samples are re-dissolved in a small amount of methanol, (2-3ml), then water, (approx 10ml), is added, and the sample dried in the Genevac HT-24 (Figure 3) using vacuum ramping (Dri-Pure) to prevent bumping and full vacuum to freeze the water. Or; samples are re-dissolved in a small volume of tertiary butanol and then dried at full vacuum. Tertiary butanol freezes at +25°C and therefore lends itself to lyophilisation very well.

Though these methods do not always deliver a light "fluffy" solid the resultant crystalline sample is both dry and weighable.  Theoretically it would be possible to lyophilise the HPLC fractions straight from the fraction collector, however this would require power handling to pool fractions, and this does not lend it self well to automation.

Using this new methodology previously difficult to dry samples are delivered in a totally dry, easy to manipulate format. By using their existing Genevac HT-24 centrifugal evaporator the need for additional laboratory equipment, such as a freeze drier, was eliminated. Developing this new methodology has enabled KuDOS to deliver full dry, weighable samples to their colleagues at AstraZeneca.