Supplementary Figure 1: Base Peak Intensity (BPI) Chromatogram for Injections of Diluted

Supplementary Figure 1: Base Peak Intensity (BPI) chromatogram for injections of diluted male and female rat urine using linear and G5 gradients, to illustrate increased resolution of peaks

Supplementary Figure 2: Multiple time series plots showing the effect of gradient and run time on peak integrals for phenylacetylglycine indiluted female rat urine

Supplementary Figure 3: Multiple time series plots showing the effect of gradient and run time on peak integrals for hippurate inartificial urine

Supplementary Figure 4: Multiple time series plots showing the effect of gradient and run time on peak integrals for hippurate indiluted female rat urine

Supplementary Figure 5: Multiple time series plots showing the effect of gradient and run time on peak integrals for phenylalanine inartificial urine

Supplementary Figure 6: Multiple time series plots showing the effect of gradient and run time on peak integrals for phenylalanine indiluted female rat urine

Supplementary Figure 7: Multiple time series plots showing the effect of gradient and run time on peak integrals for creatine inartificial urine

Supplementary Figure 8: Multiple time series plots showing the effect of gradient and run time on peak integrals for creatine indiluted male rat urine

Supplementary Figure 9: Multiple time series plots showing the effect of gradient and run time on peak integrals for creatine indiluted female rat urine

Supplementary Figure 10: Multiple time series plots showing the effect of gradient and run time on peak integrals for methionine inartificial urine

Supplementary Figure 11: Multiple time series plots showing the effect of gradient and run time on peak integrals for methionine indiluted male rat urine

Supplementary Figure 12: Multiple time series plots showing the effect of gradient and run time on peak integrals for methionine indiluted female rat urine

Supplementary Figure 13: Multiple time series plots showing the effect of gradient and run time on peak integrals for citrate (Na+ adduct) inartificial urine

Supplementary Figure 14: Multiple time series plots showing the effect of gradient and run time on peak integrals for citrate (Na+ adduct) indiluted male rat urine

Supplementary Figure 15: Multiple time series plots showing the effect of gradient and run time on peak integrals for citrate (Na+ adduct) indiluted female rat urine

Supplementary Figure 16: Plot of retention time behaviour of hippurate in diluted male and female rat urine. Shifts in retention time across gradients and time are illustrated, with reproducibility of single gradient/run time combinations clearly observed

Supplementary Figure 17: Plot of peak width behaviour of hippurate in diluted male and female rat urine. Changes in peak width across gradients and time are clearly illustrated

Supplementary Figure 18: Plot of ppm error for the accurate mass measurement of hippurate (m/z 180.0660) in diluted male and female rat urine. No definitive trend in ppm error is observed across time or gradient for this analyte

Supplementary Figure 19: Overlaid extracted ion chromatograms for the six sentinel metabolites, using gradient 5 for 8 minutes on (A) a BEH C18 column and (B) a BEH Shield RP18 column, to illustrate the differences in selectivity and peak area

Supplementary Table 1:List of compound standards, their concentrations in Artificial Urine (AU) and their final concentrations after dilution with HPLC-grade water in the ratio 1:3 AU:water

Standard / Original Concentration in AU
(mM) / Final Concentration in dilute AU
(mM)
Formic Acid / 1 / 0.25
Trimethylamine-N-oxide / 0.5 / 0.125
Alanine / 0.2 / 0.05
Lactic Acid / 0.5 / 0.125
Valeric Acid / 0.2 / 0.05
Caproic Acid / 0.5 / 0.125
Valine / 0.2 / 0.05
Taurine / 0.2 / 0.05
Creatine / 1 / 0.25
2-oxoglutaric Acid / 1 / 0.25
Methionine / 0.2 / 0.05
Ribose / 0.5 / 0.125
N-acetylcysteine / 0.2 / 0.05
Phenylalanine / 0.2 / 0.05
Uric acid / 0.5 / 0.125
Arginine / 0.2 / 0.05
Hippuric Acid / 1.5 / 0.375
Glucose / 1.5 / 0.375
Citric Acid / 1.5 / 0.375
Phenylacetylglycine / 0.2 / 0.05
Phosphocreatine / 0.5 / 0.125
Indoxylsulphate / 0.2 / 0.05
Maltose / 0.5 / 0.125

Supplementary Table 2:Summary table providing an outline of the 42 elution profiles (6 gradients over 7 run times) for UPLC-MS analysis of urine

Table header text refers to run times (in minutes) and thus points during the chromatographic run where a gradient profile change occurs. Table body text refers to gradient profiles and the corresponding percentage of solvent A at that point, where solvent A = water with 0.1% formic acid (solvent B in all cases = acetonitrile with 0.1% formic acid)