13. A New Approach to the Analysis of Hydrophilic Metabolites in Clinical Samples

Mon 12:06 PM - PosterSplash Track 2

Joseph Pesek San Jose State University

Joseph Pesek, San Jose State University Maria Matyska, Microsolv Technology Corporation John Duley, University of Queensland Steven Fischer, Agilent Technologies Theodore Sana

The determination of hydrophilic metabolites is a crucial analytical problem in clinical laboratories where new methods are in demand. Current technology involving HPLC utilizes columns that perform poorly with respect to reproducibility, ruggedness and the ability to analyze a broad range of metabolites. Silica hydride columns have demonstrated superior performance for a broad range of hydrophilic metabolites. The method used for these analytes is referred to as Aqueous Normal Phase (ANP) chromatography. The concept will be discussed and some general features with respect to metabolites presented. More specific examples related to the analysis of nucleotides for clinical analysis will be discussed. An ANP HPLC method coupled with UV or electrospray ionization (ESI)/ mass spectrometry detection (MS) was used for the determination of a wide variety of nucleotides, essential in metabolomics studies. The chromatographic conditions were changed to achieve optimized retention and separation of the nucleotides studied. The ANP-HPLC methods were developed utilizing a Diamond Hydride (Cogent) or an undecanoic acid (UDA) column, with UV detection or with an ESI MS system. Volatile, low ionic strength mobile phases were used. A negative ion mode ESI-MS at near neutral pH mobile phase was used to improve the sensitivity of the developed method. The protocol was tested with a Time-of-Flight (TOF) detector but is equally suitable for quadrupole and ion trap instruments. A simplified MS-compatible separation for UDP-glucose and UDP-galactose, to measure levels in galactosemic patients was then developed. This determination is a more challenging analysis since it involves the separation of two nucleotide-sugar esters having the same molecular weight. When the DH column was used the desired separation between these two molecules was achieved in real patient samples and in addition all other nucleotides eluted after these two compounds and did not interfere with the analysis. Further examples of clinically-related metabolite analyses will also be presented

Email: pesek@sjsu.edu

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