Introduction

1. (A) P2 (A) Metabolite Profiling in Human Urine by NMR and MS Mulu Gebremedhin1,2, Lisa Nikolai1,2, Godwin Amegbey2, David Wishart2,3 and Liang Li1 Departments of 1Chemistry, 2Biological Sciences, and 3Computing Science, University of Alberta, Edmonton, Alberta, Canada Overview Results and Discussion Experimental Six independent HPLC experiments were performed to increase the concentration of the metabolites for NMR analysis. The extracted urine sample was injected (5 mL each) onto a C18 column at a flow rate of 5 mL/min using 0.1% TFA in acetonitrile and the UV-chromatograms are shown in Figure 2.  Metabolites were separated by 1D HPLC (250 x 10 mm C18 column) using 0.1% TFA in ACN • The fractions were subjected to speed vac concentrator to remove acetonitrile and then lyophilized. • The flow chart of the extraction is shown in Figure 1 In this work , MS data were collected for all the pooled fractions at both positive and negative ion mode to determine the molecular mass of the metabolites which assists the interpretation of NMR spectra for metabolite identification. Overall, 70 fractions were collected from each experiment and subjected to speed vac to remove acetonitrile and pooled into 13 fractions based on retention time and then freeze-dried and lyophilized overnight. The dried pooled fractions were dissolved in solvents compatible with NMR and internal standards were added according to the standard operating protocol and analyzed by 500 MHz NMR as shown in Figure 3. Purpose • To identify and quantify metabolites from human biofluids using MS and NMR Method • 1D-HPLC coupled to MS and NMR Results  A total of 48 metabolites were identified and quantified from a normal human urine sample (B) Example of MS Search Results Metabolite Extraction and Fractionation ACN Centrifuge Urine Figure 4. (A) MS spectrum (positive mode) of pooled fraction 2 obtained from the single quadrupole mass spectrometer by flow injection of 2 L solution. (B) MS search results for a single m/z value (136.1) against a Human Metabolome Database ( www.hmdb.ca) Introduction (1:1) The determination of all possible metabolites from urine is important in the study of human diseases. GC and GC-MS have been widely used for the analysis of specific metabolites in urine. However, the sample preparation for GC and GC-MS is time consuming, because derivatization procedure is often required. Sample preparation for HPLC is relatively simple. In this work we have developed a method to isolate, purify, identify and quantify metabolites in urine by HPLC-MS and NMR. Table I. Partial list of identified and quantified human urine metabolites obtained using NMR Chenomx eclipsed software. The reported concentration is from the Human Metabolome Database Remove Supernatant ACN (B) P3 Original volume Fractionation by HPLC (C ) P4 Experimental Instrumentation • Single quadrupole mass spectrometer (Agilent HP 1100 MSD ) • 500 MHz NMR (Varian Unity Inova) • HPLC system (Beckman Courier) Sample Preparation and Extraction • Urine sample was collected in a sterile container from a healthy volunteer • Acetonitrile (ACN) and aqueous methanol were used to extract the metabolites • The supernatant were subjected to rotary evaporation at room temp. Conclusions and Future Work • The MS data were used to generate a list of possible metabolites based on the molecular mass match with those listed in a Human Metabolome Database. • NMR was used to identify the metabolites. A total of 48 compounds were identified by HPLC-NMR. • Most of the metabolites identified in this work are high abundance. In the future, 2D-HPLC will be used to enrich low abundance metabolites. • We will also focus on identifying novel metabolites using 2D-HPLC-FT-MS and NMR. (D) P5 Pictures of some urine fractions Figure 1. Flow chart of metabolite extraction protocol (only with ACN), fractionation by HPLC and picture of urine fractions collected from reversed phase HPLC. Figure 2. The first four UV-chromatograms of the reversed phase HPLC separation of the extracted metabolites from normal human urine. Figure 3. Typical examples of the NMR spectra for pooled fractions 2(A), 3(B), 4(C) and 5(D). Water and DSS (at 0.0 ppm) peaks are removed from the spectra. Acknowledgements This work was funded by Genome Alberta and Genome Canada through the Human Metabolomics Project.