Investigation of the separation mechanism in hydrophilic interaction chromatography.

Lead Research Organisation: University of the West of England
Department Name: Faculty of Health and Applied Sciences

Abstract

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.

Planned Impact

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.
 
Description Our work has contributed extensively to knowledge concerning the mechanism of hydrophilic interaction chromatography (HILIC). We have discovered that the most important parameters for controlling the selectivity of the separation are the nature of the stationary phase and the mobile phase pH, the latter is clearly applicable only for ionisable compounds. Stationary phases such as amide or zwitterionic can trap considerable layers of water into which analytes can partition. Peak shape is determined by the ionic strength of the mobile phase which varies considerably in high concentrations of organic solvents as used in HILIC compared with the situation in water. Thus we found that formic acid containing mobile phases give poor results. Salt buffers such as ammonium formate and ammonium acetate should be used instead. The kinetic performance of HILIC compared with reversed-phase was surprising in that high diffusivity of sample molecules is expected in HILIC mobile phases. This should lead to high B terms and small B terms in the Van Deemter equation. Practically, we established that the reverse is true probably due to reduced diffusivity in water layers close to the surface.
Exploitation Route We have already shown that HILIC is an important technique in the pharmaceutical industry for assessing impurities and for assays of polar drugs. It is also excellent for monitoring antibiotics, many of which are hydrophilic and cannot be analysed by conventional reversed-phase techniques.
Sectors Chemicals

Environment

Healthcare

Pharmaceuticals and Medical Biotechnology

 
Description We studied the mechanism of hydrophilic interaction chromatography, and have shown how the separations can be manipulated to give the optimum performance. We have worked closely with the pharmaceutical company GlaxoSmithKline to implement these findings such that they have additional screening methods for determining (in particular) polar impurities in their products. It is a regulatory requirement that impurities should be present at levels of less than 0.1% (or 0.05 %) of the active pharmaceutical ingredient. Some of our methods have already been implemented by GlaxoSmithKline in routine procedures. We have also transferred some of our methodology to the Antibiotics Reference Laboratory in Bristol, giving rise to improved methods for the analysis of these substances. Many antibiotics are polar compounds much more suited to hydrophilic interaction chromatography than reversed-phase techniques. Our methods are being used to determine circulating levels of these compounds in body fluids. The more theoretical aspects of our work have also allowed a greater understanding of this technique in the general research community. There is much interest in the technique as evidenced by the considerable number of invited lectures delivered nationally and internationally by the Principal Investigator over the period of the funding.
First Year Of Impact 2013
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description QR funding
Amount £25,000 (GBP)
Organisation Higher Education Funding Council for England 
Sector Public
Country United Kingdom
Start 11/2014 
End 06/2015
 
Description Agilent Technologies Instrument Development 
Organisation Agilent Technologies
Country United States 
Sector Private 
PI Contribution We have investigated features of instrument design of ultra-high performance liquid chromatographs. We have developed a method to measure coefficients representing the diffusion of organic compounds through packed beds of separation material.
Collaborator Contribution Agilent Technologies have provided state of the art ultra performance liquid chromatography equipment for use in this work.
Impact Publication "Practical observations on the performance of bare silica in hydrophilic interaction chromatography compared with C18 reversed-phase liquid chromatography" J.C. Heaton (UWE) X.Wang (Agilent), W.E. Barber (Agilent) D.V. McCalley (UWE), J. Chromatogr. A 2014 1328 7-15.
Start Year 2012
 
Description Antiobiotic analysis using HPLC 
Organisation Southmead Hospital
Department Antimicrobial Reference Laboratory (ARL)
Country United Kingdom 
Sector Hospitals 
PI Contribution We have provided the expertise in hydrophilic interaction chromatography and developed new procedures using this technique for the analysis of antibiotics.
Collaborator Contribution The laboratory has donated time on an HPLC-ESI mass spectrometer; has purchased HPLC columns and standards on our behalf, and provided blank plasma samples for analytical recovery work.
Impact We have produced new methods for antibiotic analysis. Many of these compounds are hydrophilic and not retained by conventional methods in reversed-phase high performance liquid chromatography, but are easily analysed by our new methods in hydrophilic interaction chromatography (HILIC) . We are looking at the reproducibility of these HILIC methods, particularly the establishment of quasi equilibrium with the mobile phase, which gives satisfactory analytical reproducibility without the time penalties that result from waiting for a full equilibrium.
Start Year 2012
 
Description Pharmaceutical analysis with GSK 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution UWE has developed HPLC methods for pharmaceutical impurity detection that have been used by the company for screening procedures for these compounds.
Collaborator Contribution Provided pharmaceutical standards and HPLC columns for the research.
Impact Publication: J.C. Heaton (UWE), J.J. Russell (UWE), T. Underwood (GSK), R.Boughtflower (GSK), Comparison of peak shape in hydrophilic interaction chromatography using acidic salt buffers and simple acid solutions, J. Chromatogr. A 2014 1347 39-48
Start Year 2012
 
Description Industrial meeting, Munich Germany 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Almost 50 industrialists and instrument designers met to discuss improvements in the design of ultra-high performance liquid chromatography instruments. I gave a lecture on the consequences of the use of short, narrow columns for fast separations on the design of instruments. Factors such as the bandspreading contribution from the injector, detector and connecting tubing were discussed, along with the consequences of adiabatic and isothermal heating systems on separation performance.

Decisions were debated on facilities to include in new designs of instruments.
Year(s) Of Engagement Activity 2014
 
Description Keynote Lecture and Lecture HPLC 2014 (St Louis, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a keynote invited lecture to an audience of about 400 at International HPLC conference on the results of this EPSRC project. The talk covered influence of true thermodynamic pH on retention in hydrophilic interaction chromatography.

Postdoctoral worker employed on the project compared the kinetic performance of superficially porous and totally porous packings, looking particularly at the peak parking method for measruing diffusion within the column.

There was considerable discussion of these papers after the presentations.

Invited to participate in meeting to influence future instrument design in Munich later in 2014.
Year(s) Of Engagement Activity 2014
 
Description Keynote invited lecture, Separation Science Conference 2014 (Salzburg, Austria) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact About 400 industrialsts and academics attended this keynote lecture entitled "Hydrophilic Interaction Chromatography-the state of the art" in Salzburg Austria. This resulted in further discussions with instrument manufacturers on the design of instruments.

Invited to apply for a research gift from Agilent Technologies in USA to develop methods further for the analysis of drugs.
Year(s) Of Engagement Activity 2014
 
Description Keynote lecture HPLC 2013 Conference (Amsterdam, Netherlands) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Keynote lecture on new approach to analysis of pharmaceuticals. Stimulated interest from pharmaceutical companies,

Asked to visit industrial company to encourage cooperation in this area,
Year(s) Of Engagement Activity 2013
 
Description Public lecture on application of analytical techniques in modern life 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public lecture given to non-specialist audience.

This was one of a series of public lectures organised by the university to communicate ideas and applications of current research projects to the general public.

Audience reported real interest in how these techniques had been used in health science and forensic science.
Year(s) Of Engagement Activity 2014