Investigation of the separation mechanism in hydrophilic interaction chromatography.

Chromatography is a method of separating and analysing important chemicals such as ethical pharmaceuticals, drugs of abuse, food additives, pesticides and compounds in biological fluids that may be indicative of disease. A popular variant is high performance liquid chromatography, where the sample is introduced into a liquid stream (the "mobile phase") and passed through a column containing particles of the separation medium (the "stationary phase"). If these particles are made smaller, then the separations improve and they can be accomplished in minutes or even seconds, rather than hours or days. Small particles however, require high pressures to force the liquid through, hence the term high performance (or high pressure) liquid chromatography (HPLC). A detector situated at the end of the column senses the separated chemicals and allows them to be quantified. The annual world market for instruments and consumables in HPLC is several billion dollars; tens of thousands of analyses are carried out daily in the UK alone. Thus, faster and more efficient variations of the technique are continually being sought. One of these is hydrophilic interaction chromatography (HILIC), a technique that has significant advantages over the commonly used separation procedure (reversed-phase, RP), particularly for the fast analysis of drugs and compounds of clinical significance. Many of these compounds have only weak interactions with the usual RP columns and thus give poor separations. However, the mechanism of HILIC is poorly understood, which makes it difficult to design the separation conditions. Some separations seem to work very well, whereas others do not, for no apparent reason. The aim of this research is to elucidate the principles of the HILIC separation mechanism and to elucidate the experimental parameters that are most influential on the separation. This knowledge will be used to develop a "tool box" approach to apply the technique to separations that have proved difficult by conventional techniques. The idea is to apply an automated series of HILIC separations using different experimental conditions that generate different selectivity, such that an optimum for the analysis can be chosen. The study is of particular importance to the pharmaceutical industry especially in connection with new methods of drug design, such as "fragment based drug discovery" which involve synthesis of more complex structures using small building blocks each containing some desirable property of the final compound. Many of these building blocks are small polar molecules that should be very well suited to the HILIC technique. All pharmaceuticals need to be carefully screened for the presence of impurities, that may give rise to undesirable physiological side effects. HILIC can be used for example to assess the presence of residues of these polar starting materials in the final product. HILIC appears less susceptible to "overload", a phenomenon where distortion of the separation takes place when larger amounts of material are analysed. Overload can cause interference between large peaks of the active pharmaceutical ingredient and small quantities of impurities.
The proposed work will require the development of a mixture of compounds of different structure that can be used to probe the various contributions to retention in HILIC. While the HILIC technique is very simple in practice, it appears that there are many complex factors which contribute to the separations. For example, many important pharmaceuticals, drugs of abuse, and compounds of biomedical significance are charged species, which adds an extra dimension of complexity to the separation mechanism. Understanding the importance of these various contributions will allow separations to be designed which can fully exploit the useful differences between HILIC and conventional separation methods.

Who will benefit from this Research?
1) The UK Pharmaceutical industry through the development of more efficient methods of analysis.
2) The Separation Science and Analytical Chemistry community within the U.K. in contributing to training and development of valuable skills in this area.
3) Hospital chemistry laboratories through the development of simpler methods for the analysis of biomarkers in body fluids.
4) Manufacturers of chromatographic columns and instrumentation for performing HPLC.
5) The general population through improvements in assessment of food and health products, and improvements in environmental monitoring.

How will they benefit from this research?
1) Pharmaceutical companies continually search for faster, cheaper and more efficient ways of analysing and assessing raw materials and finished products.. The pharmaceutical industry is a huge contributor to the U.K. Economy, with a trade surplus >£ 5 billion annually. The value of pharmaceutical exports in 2007 was around £15 billion. A considerable amount of work with HPLC is carried out. For example, impurities that can potentially have harmful side effects, are of major concern. HILIC is potentially an ideal method for assessing impurities, and residues of small polar molecules in techniques like fragment-based drug discovery. An understanding of the mechanism of HILIC would improve these assays which are very difficult to perform using conventional techniques. Reporting of impurities that represent around 0.05% of the API is now a legal requirement. A better understanding of the HILIC process would lead to improvements in the design of columns and separation for such analyses.

2) While Separation Science plays a major role in the health and well-being of the entire UK community, research has declined over the past two decades in the U.K. Belgium, a country with only a sixth of the U.K. population, has a strong academic presence in the subject, with two academic chairs advertised over the past 6 months alone that will complement at least 5 other similar positions. In contrast, there are no permanent academic chairs in Separation Science in the UK. The award of this grant would give strong encouragement to those few U.K. researchers in this subject.

3) Many compounds of biomedical and clinical interest are strongly polar hydrophilic substances. These compounds can present severe difficulties for analysis due to their poor peak shape and poor retention on conventional reversed-phase columns. The applicant has been recently looking at the application of HILIC to catecholamines, which are routinely determined by HPLC in hospital laboratories. Substitution of a HILIC procedure has the potential for improving the speed and quality of these analyses, which will benefit the diagnosis of tumours such as phaeochromocytoma and neuroblastoma.

4) The applicant has considerable contacts with column and instrument manufacturers, who have donated state-of- the- art instruments for his research programme (Agilent 1290 UHPLC, Waters Acquity UPLC) of total value around £150,000. The research should result in information concerning new types of column and methods for their use to solve common analytical problems. It should lead to the development of new column types suitable for HILIC.

5) Separation Science is an important contributor to the health and well-being of the population of the U.K. It is also used for the determination of pesticides and other contaminants in foodstuffs, and for environmental monitoring. While much of this work is routinely performed in industry, little research is performed to make fundamental improvements.

It is expected that improvements in methodology resulting from this project could be implemented in pharmaceutical and hospital laboratories in 2-3 years.