Alan McKeown (Presenter)
Advanced Chromatography Technologies Ltd
Bio: Alan P McKeown BSc, PhD, CChem, FRSC is the Business Development Director for Advanced Chromatography Technologies Ltd, UK. Alan is both commercially and technically focussed working with multiple functions at ACT that include R&D / new phase design, Sales, Marketing and Technical Support. Alan also directly works with customers for chromatography support, troubleshooting and method devlopment. As an experienced chromatographer, Alan has been invited and delivered chromatography technical seminars in Europe, Asia, North America and South America at conferences, customer sites and organised events.
Authorship: Alan P McKeown (1), Catherine Ortori (2) and Geoff Faden (3)
(1) Advanced Chromatography Technologies Ltd, 1 Berry St, Aberdeen, Scotland, AB25 1HF UK (2) School of Pharmacy, The University of Nottingham, University Park, Nottingham, NG7 2RD UK (3) MACMOD Ana
| Short Abstract Profiling urine can provide useful information to aid clinical diagnosis. The use of UHPLC and High Resolution Accurate Mass (HRAM)instrumentation in clinical laboratories has grown considerably in recent times. Here, a novel C18 based polar embedded column (ACE Excel 1.7um C18-Amide) is used. This unique stationary phase has been designed to retain and separate a range of polar and non-polar species. Using an 8 minute broad gradient with HRAM it was possible to demonstrate the profiling capability of the method for a wide range of naturally occurring small polar molecules in urine. |
Long Abstract
Introduction
Human urine samples tend to be complex with a range of polar analytes from low to mid molecular weight (<100 Da to ~500 Da) present within a highly polar matrix. Profiling urine can be helpful for diagnosing the presence and progression of a variety of disease states as well as monitoring endogenous metabolic processes. Human urine presents an interesting separation and detection opportunity that can be addressed with the growing popularity of UHPLC and High Resolution Accurate MS (HRAM) within clinical laboratories. In this work a novel C18 based polar embedded column (ACE Excel 1.7um C18-Amide) is used to analyse urine from a healthy volunteer. This unique stationary phase has been designed to retain a range of polar and non-polar species but with low phase bleed making it ideal for low level work in MS applications.
Methods
Sample preparation was simple. 1ml aliquots of urine were centrifuged at 4C at 13000rpm in microfuge vials and decanted into LC vials. Samples were injected directly onto the UHPLC-HRAM system. The detector, an Exactive MS system from ThermoScientific (Hemel Hempstead, UK), was employed to monitor and identify metabolites by extracting high resolution ion chromatograms. Analyte separation was provided by the novel ACE C18-Amide (1.7um particle size). This column chemistry has been designed to contain multiple modes of interaction to provide resolution of small structurally similar polar and non-polar analytes. Various healthy volunteer urine samples were prepared and analysed.
Results
Initial method development allowed the rapid determination of conditions for retaining and separation hydrophilic and some hydrophobic small molecules within a reasonable run time. Using a final method that comprised an 8 minute broad gradient with HRAM, it was possible to profile urine samples and identify a number of analytes that included hypoxanthine (and isobarics), tryptophan and hippuric acid along with a wide range of small polar molecules.
Conclusions
A generally applicable UHPLC-HRAM urine profiling method using the novel ACE Excel 1.7um C18-Amide is described.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | ADVANCED CHROMATOGRAPHY TECHNOLOGIES LTD |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |