Systematic Isolation and Structure Elucidation of Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling Laboratory

Annotation and identification of metabolite biomarkers is critical for their biological interpretation in metabolic phenotyping studies, presenting a significant bottleneck in the successful implementation of untargeted metabolomics. Here, a systematic multistep protocol was developed for the purification and de novo structural elucidation of urinary metabolites. The protocol is most suited for instances where structure elucidation and metabolite annotation are critical for the downstream biological interpretation of metabolic phenotyping studies. First, a bulk urine pool was desalted using ion-exchange resins enabling large-scale fractionation using precise iterations of analytical scale chromatography. Primary urine fractions were collected and assembled into a “fraction bank” suitable for long-term laboratory storage. Secondary and tertiary fractionations exploited differences in selectivity across a range of reversed-phase chemistries, achieving the purification of metabolites of interest yielding an amount of material suitable for chemical characterization. To exemplify the application of the systematic workflow in a diverse set of cases, four metabolites with a range of physicochemical properties were selected and purified from urine and subjected to chemical formula and structure elucidation by respective magnetic resonance mass spectrometry (MRMS) and NMR analyses. Their structures were fully assigned as tetrahydropentoxyline, indole-3-acetic-acid-O-glucuronide, p-cresol glucuronide, and pregnanediol-3-glucuronide. Unused effluent was collected, dried, and returned to the fraction bank, demonstrating the viability of the system for repeat use in metabolite annotation with a high degree of efficiency.

S-2 1 H COSY NMR spectrum of Feature A; Figure S7 shows the 1 H-13 C HMBC NMR spectrum of Feature A; Figure S8 Extracted ion chromatograms (EIC) of the m/z 350.088 and the co-eluting feature m/z 187.007; Figure S9 Extracted ion chromatograms (EIC) and MS/MS spectra of features with m/z 350.088 (ES-); Figure S10 shows an ES-MRMS with an isotopic fine structure confirmation of elemental composition (C 16 H 16 NO 8 ) of purified Feature B; Figure S11 shows evidence of the degradation of purified Feature B when stored in phosphate buffer; Figure S12 shows the 1 H-1 H COSY NMR spectrum of Feature B; Figure S13 shows the 1 H-13 C HMBC NMR spectrum of Feature B; Figure   S14 Negative MS/MS spectrum and statistical heterospectroscopy analysis of feature C; Figure S15 shows EIC chromatogram (ES-) comparing urine pool with analytical standard of pregnanediol-3glucuronide; Figure S16 shows an ES-MRMS with an isotopic fine structure confirmation of elemental composition (C 27 H 43 O 8 ) of purified Feature C; Figure S17 shows the 1 H-1 H COSY NMR spectrum of Feature D; Figure S13 shows the 1 H-13 C HMBC NMR spectrum of Feature D; S-3

Secondary and tertiary isocratic fractionation
To generate the library of isocratic chromatography conditions, four reversed-phase C18 chemistries were assessed (Waters X-Bridge BEH C18; Waters X-select CSH C18, Waters, Sunfire C18, Waters Atlantis T3 C18)). Columns of dimensions 4.6mm x 150mm,3µm were used in each case. Two solvent combinations were employed: the first consisted of water (0.1% formic acid) and acetonitrile (0.1% formic acid), whilst the second replaced acetonitrile with methanol as the organic modifier. Overall, eleven sets of reversed phase isocratic conditions were assessed using two solvent gradients (99% A, 95% A and then at 5% interval decreases to 50% A).
It should also be noted that an additional Waters X-Bridge BEH C8 column with selectivity similarities with the X-Bridge C18 was used in cases where the X-Bridge C18 required further bespoke modification.
Two HILIC phase chemistries were additionally assessed for the database (Waters X-Bridge HILIC; Waters Atlantis HILIC). The column dimensions were 4.6mm x 150mm, 3µm in each case. For the HILIC database only one mobile phase combination was employed consisting of 20mM ammonium formate in water (A) and 0.1% formic acid in acetonitrile (B). Ten sets of HILIC isocratic conditions were assessed (5% A and then at 5% interval increases up to 50% A).

MRMS analysis
Magnetic resonance mass spectra (MRMS) were acquired with a Bruker solariX 2xR (Bruker Daltonics, Billerica, MA, US) using electrospray ionization (ES) and direct infusion with syringe pump. The fraction samples were diluted 1:9 in 50% acetonitrile + 0.1% formic acid for ES+. Fraction samples were diluted 1:9 in 50% acetonitrile ES-. Samples were measured with direct infusion using a flow of 2 µl/min. Mass spectra were acquired with a mass resolution of 1.350.000 at m/z 200 using quadrupolar detection. 64 single scans were added for the final mass spectrum. Spectra were externally mass calibrated with NaTFA cluster.

singlet (s); doublet (d); triplet (t); multiplet (m); triplet of doublets (td); doublet of triplets (dt)
S-9 encircled key correlations corresponding to the structure and assignment of Feature A as presented in Table S2.
Labels match the chemical groups as labelled in Figure 2. Figure S7. Plots presenting two details of the 1 H-13 C HMBC spectrum a) 7-8 ppm, and b) 3.1-3.7 ppm regions with encircled key correlations corresponding to the structure and assignment of Feature A as presented in Table   S2. Labels match the chemical groups as labelled in Figure 2.

S-19
Figure S12. Plots showing two details of the 1 H-1 H COSY spectrum a) 6.8-8 ppm, and b) 3-5.5 ppm regions with encircled key correlations corresponding to the structure and assignment of Feature B as presented in Table S3 in the main text. Labels match the chemical groups as labelled in Figure 2.
Figure S13. Plots presenting two details of the 1 H-13 C HMBC spectrum a) 7 -7.8 ppm, and b) 3.5-4.1 ppm regions with encircled key correlations corresponding to the structure and assignment of Feature B as presented in Table   S3 in the main text. Labels match the chemical groups as labelled in Figure 2.
S-20   Table S4. Labels match the chemical groups as labelled in Figure 2.  Table S4. Labels match the chemical groups as labelled in Figure 2.