Pharmaceutical analysis in drug development

It is clear that pharmaceutical analysis plays a very important supporting role in drug development. Already during the conception of a candidate drug, for example by chemical synthesis, suitable analytical means are required to determine the identity and purity of the compound. Subsequent studies performed on candidate drugs with the aim of establishing the pharmacological, toxicological and therapeutic characteristics of the candidate drug vastly rely on bioanalysis.

Although there is still an important place for wet chemistry analysis such as, for example, identification tests, limit tests, titrations for assay etc. in the quality control of bulk drugs, the trend is in many cases to shift to separation techniques because the resolution they offer gives enhanced information about the sample.

In the pharmaceutical industry most laboratories are mainly equipped with liquid chromatography (LC) as a separation technique, coupled to diode array detection or mass spectrometry (MS). This technique separates the compounds in a mixture based on differences in interaction with the stationary phase, be it hydrophobic interactions, affinity interactions or separations based on size of the molecules. LC has experienced such tremendous success because the technique is able to yield analytical results in a repeatable manner. Since it has been around for a long time, the analysts have learnt many aspects of it, so that troubleshooting has become more obvious. LC is quite robust which makes life easier when methods need to be transferred from one company site to another. Moreover, the development of LC has never stopped, in the sense that stationary phases have been continuously improved by the column manufacturers with new phases coming on the market for highly polar compounds for instance, or for basic compounds that tend to show peak tailing. There is currently also better knowledge on the differences in selectivities of stationary phases and the underlying chemistries. Another significant advance has been the miniaturisation at the level of column size as well as particle diameter. Both allow the analyst to obtain better results because the efficiency of the separation improves; less sample is needed; the mass sensitivity increases and less reagents are needed to do so, which is advantageous for environmental and economical reasons.

Gas chromatography (GC) was the first chromatographic technique to be developed and it is still very useful for analysing volatile drugs. The majority of drugs are, however, non volatile and the focus of GC lies in the analysis of residual solvents. These can remain in a finished product after having been used during the production process. Residual solvents vary in their toxicity and therefore the limits allowed in drugs depend on the type of residual solvent. GC enables the determination of the level of residual solvents present in any bulk drug or finished preparation.

Companies also still use thin layer chromatography (TLC) in chemical laboratories, exploiting the fact that it provides information quickly on the composition of a mixture during the synthesis of a new chemical entity. Densitometric equipment allows quantitative data to be obtained concerning samples, but in this respect TLC is being overruled by LC, which performs better in quantitative work. Very complex mixtures such as plant extracts and herbal drugs benefit from the ease of use of TLC and its large variety of possible revelation modes, and are thus often analysed by TLC.

It is widely considered that in the future there will be greater focus on the hyphenation of separation techniques with mass spectrometry because such systems enable structural information to be obtained on all separated compounds (impurities, metabolites etc.). LC/MS is now omnipresent in pharmaceutical companies and new developments such as Ultra Performance Liquid Chromatography (UPLC) coupled to MS have been introduced. UPLC/MS sees an increase of efficiency and speed of analysis due to the drastic decrease in particle size of the stationary phase. Other coupled techniques with high level of significance are TLC coupled to MS with the use of Matrix Assisted Laser Desorption Ionization and Time of Flight detection (MALDI-TOF) or Supercritical Fluid Chromatography coupled to MS (SFC/MS).

In some cases the situation can arise that not enough resolution is obtained from these chromatographic interactions, in which case the analyst looks for other separation techniques based on different separation mechanisms.

One such technique, which has recently been developed, is capillary electrophoresis (CE). This is where a high voltage is applied onto a tube of capillary dimensions, filled with a background electrolyte and dipped at each side into vials filled with electrolyte. The sample is introduced into the capillary and under the influence of the electric field the compounds in the mixture start migrating. If the mobility of the distinct constituents of the mixture is different, they will all reach the detector at different times and separation is observed. Since the separation is in this case based on a different mechanism, CE can offer complementary separation with LC, so that a combination of the two techniques in the laboratory can give additional information about a sample. CE also offers many different types of selectivities, because depending on the solution with which the capillary is filled, one can focus on charge to mass ratio separation, or on separations based on differences in hydrophobicity between the compounds, or based on different sizes or pIs. Given the strong pressure currently placed on analytical labs due to economical reasons, lab managers often choose to have this additional technique in house. Indeed, the time gain that is often obtained by using CE, as well as the lower solvent and reagent consumption in this miniaturised technique, allows the analyst to obtain results faster and at a lower cost.

Up until now, capillary electrophoresis has not found the same widespread use in industry as LC- probably due to the fact that technological advances and equipment manufacture have come after LC and that the latter technique is performing quite well. In addition some companies have experienced a lower repeatability with CE than with LC, as well as occasional problems of method transfer between sites. On the other hand, CE has proven its advantage for particular applications, such as in chiral separations. The large amount of chiral selectors available, comprising neutral as well as charged and chargeable cyclodextrins, enables highly efficient separations between enantiomers to be obtained – often with minimal development time. This avoids the laboratories the purchase of expensive LC columns for chiral separation, of which the good performance for a particular application cannot be guaranteed in advance. Another field in which CE is certainly advantageous is the analysis of therapeutic proteins, and the implementation of CE by pharmaceutical industry in texts for regulatory purposes is increasing with time. CE can, for example, be very useful to determine the charge heterogeneity of glycosylated proteins, which is usually typically determined by iso-electric focusing in flat gels. In addition, the various structural modifications that a protein can undergo during preparation and storage, need to be thoroughly monitored when constituting a registration file. CE can give as good results in this field as LC, so companies often use CE for this purpose, in view of its lower consumable expenses. The analytical laboratories should keep in mind, however, that it is important to offer sufficient training to the analysts. CE is somewhat less incorporated in traditional analytical education and many analysts approach the technique from their background knowledge as a chromatographer. The underlying mechanisms are however different and in many cases good training on a particular type of equipment is essential for successful operation. A typical example is the elaboration of CE-MS methods. The coupling of capillary electrophoresis equipment to a mass spectrometer yields very powerful results, on condition that the analyst receives enough support and troubleshooting advice from the instrument manufacturers.

In conclusion, it can be emphasised that capillary electrophoresis has the potential to be a real ‘problem solver’ for analytical questions that are sometimes difficult to answer with the aid of other analytical techniques. The variety for separation selectivities together with very high efficiencies, low analysis times and low reagent consumption makes it a separation technique that should always be considered when developing methods for quality control of drugs, or when establishing regulatory texts for drug candidates.

A. Van Schepdael

Laboratory of Pharmaceutical Analysis, K.U.Leuven

Ann Van Schepdael obtained a degree in pharmacy, as well as a PhD in Pharmaceutical Sciences at the Catholic University of Leuven (K.U.Leuven) in Belgium. She is currently Professor and affiliated with the Laboratory of Pharmaceutical Analysis, K.U.Leuven.