John Haselden 1, Gordon Dear 1, Jennifer H. Granger 2, and Robert S. Plumb 2. 1GlaxoSmithKline, Ware, UK; 2 Waters Corporation, Milford, MA, USA

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1 Challenges of Polar Compound Analysis Routine LC/MS-based analyses usually rely on using reversed-phase column chemistries (e.g. C 18, C 8, C 4 ) in their LC methodology. However, one of the shortcomings of this approach is the inability to retain and separate compounds such as amino acids, sugars, and organic acids. It is well known that these polar compounds elute at the void volume when reversed-phase column chemistries are employed under typical LC/MS operating conditions, as illustrated by Figure 1. In the past, solutions to this dilemma have involved the use of derivatizing reagents prior to analysis, but this may require several time-intensive sample preparation steps. Recently, we have developed a new strategy for the analysis of polar compounds in urine. This LC/MS-friendly method focuses on the use of a new polymeric cation exchange column from the Atlantis family of column chemistries and a simple organo-aqueous ammonium formate based gradient. Using this approach, we have been able to show that this method can successfully resolve mixtures of amino acids, sugars, and polar neutrals in rat urine as part of an LC/MS time-of-flight (TOF) approach. Additionally, the LC/MS TOF data generated using this approach can be analyzed for metabonomic information using a chemometric algorithm such as principal components analysis (PCA). A NEW COLUMN CHEMISTRY FOR METABONOMIC ANALYSIS OF POLAR COMPOUNDS IN BIOLOGICAL MATRICES John Haselden 1, Gordon Dear 1, Jennifer H. Granger 2, and Robert S. Plumb 2 1GlaxoSmithKline, Ware, UK; 2 Waters Corporation, Milford, MA, USA Experimental Conditions Urine samples from dosed male and female Han Wistar rats were collected 0-8 and 8-24 hours post-dose. Each group received a once-daily oral dose of the candidate pharmaceutical or dosing vehicle for a total of 90 days. The dosing levels were 0 mg/kg, 2 mg/kg or 18mg/kg. LC/MS Conditions All LC/MS data was acquired using a Alliance HT 2795 XC Separations Module interfaced with a Waters Micromass Q-Tof micro Mass Spectrometer with a LockSpray Ionization Source. All resulting LC/MS metabonomics data was processed using the MarkerLynx Application Manager for MassLynx 4.0 Software. Column: Flow Rate: Injection Volume: 20 µl Waters Atlantis Metabonomics Column, 5 µm 2.1 mm x 100 mm 600 µl/min Gradient: Linear gradient, 0-95%B in 20 or 40 minutes (A = 0.1% formic acid in water and B = 50 mm Ammonium Acetate in 1:1 water:acn) Ion Mode: Voltages: Positive ion electrospray Cone, 30V Capillary, 3200V Desolvation Temp. 250 C Source Temp. 120 C Figure 1: Representative chromatogram for rat urine on a traditional reversed-phase C 18 column. Collision Energy: Dwell Time: Collision Gas: Lock Mass: 12eV 0.1sec Argon Leucine Enkephalin, 50 fmol/µl

2 Figure 2. Chromatographic behavior of representative amino acids on the Atlantis polar phase. Results Investigation of Amino Acids Prior to the analysis of polar compounds in urine, the chromatographic behavior of several amino acid standards were evaluated on the new phase. Figure 2 shows several representative total ion chromatograms for amino acid standards. In all cases, the amino acids are well retained and have reasonable peak shapes. An additional parameter investigated was the effect of the ammonium formate concentration on peak shape. The concentration was varied from 50 mm to 300 mm, and in general, it has been found that a concentration of 50 mm is optimum for most amino acids, sugars, and polar neutrals. Typical results for organic acids are presented in Figure 3. Metabonomic Analysis of Urine from Dosed Rats The aforementioned strategy for polar compounds was applied to the analysis of a set of urine samples from male and female rats dosed at two levels with a candidate pharmaceutical. The resulting chromatograph, presented in Figure 4, is for a representative male rat urine sample from control, low dose, and high dose sample groups. Although differences among all three total ion chromatograms can be observed, the subtle changes in compounds from sample to sample is best visualized using a chemometric approach such PCA or partial least squares discriminant analysis (PLS-DA). To this end, the resulting scores and loadings plots from PCA of LC/ MS data from all male urine sample from control, low dose and high dose groups are shown in Figure 5.

3 Figure 3. Exact mass extracted ion chromatograms for (a) hydroxybutyric acid, (b) 3-methylglutaric acid, and (c) pyroglutamic acid on the Atlantis polar phase. Figure 4. Total ion current chromatograms (TIC) for urine samples from control, low dose and high dose group rats using an Atlantis polar column (data has been expanded to show low abundance). Many differences between these three chromatograms can be visualized.

4 Scores: Component 1 - Component 2 ApplicationNOTE 2 t n e n o p m o C Control Low Dose High Dose Component 1 Figure 5. Resulting scores plot (left) and loadings plot (right) from PCA of LC/MS TOF data from control, low dose, and high dose rat urine from male animals on day 85 of the study. Distinct clustering by dose group is observed. The loadings plot shows that several ions, possibly polar metabolites of the candidate pharmaceutical contribute to the clustering observed in the scores plot. Figure 6. Resulting scores plot (left) and loadings plot (right) from PLS-DA of LC/MS TOF data from male and female rat urine from the high dose group on day 85 of the study. Clustering based on gender variation amongst the high dose animals is displayed.

5 Another more subtle comparison can be made for this sample set between male and female animals in a single dose group. Figure 6 displays the resulting scores and loadings plots for male and female rats from the high dose group on day 85 of the study. In this example, the peak list from MarkerLynx has been processed in Pirouette chemometrics modelling software for enhanced three-dimensional data viewing. Conclusion The LC/MS analysis of underivatized polar components in rat urine has been successfully accomplished using a new Atlantis column chemistry and an ammonium formate-based organo-aqueous mobile phase. This methodology is extremely useful in the metabonomics arena, where many polar molecules such as amino acids, sugars, and organic acids have biological relevance. The acquired LC/MS data was processed using chemometric data analysis tools, and the subsequent results presented here illustrate the utility of this new column chemistry for metabonomics. WATERS CORPORATION 34 Maple St. Milford, MA U.S.A. T: F: Waters, Micromass, Q-Tof micro, Atlantis, LockSpray, MarkerLynx, MassLynx and Alliance are trademarks of Waters Corporation. All other trademarks are the property of their respective owners Waters Corporation Produced in the U.S.A. August EN MB-PDF