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Use case · Biotransformation

Metabolite identification

Rank metabolite and conjugate hypotheses from isolated or enriched NMR datasets with explicit residual uncertainty.

Starting point
Isolated or enriched metabolite NMR from incubations or biofluids
Typical input
1H and 13C, solvent (typically D2O or DMSO-d6), plus parent structure or formula
Spectra output
Ranked metabolite and biotransformation candidates with per-shift residuals
Chemist's call
Overlay authentic standard, confirm regioselectivity by 2D, or keep provisional
Metabolite identification analytical schematic
WORKFLOW SCHEMATIC — Rank metabolite and conjugate hypotheses from isolated or enriched NMR datasets with explicit residual uncertainty.
Loading 1H, 13C, and supporting 2D acquisitions (HSQC, COSY, HMBC, TOCSY, NOESY) with parent-structure context.
IN SPECTRA — Loading 1H, 13C, and supporting 2D acquisitions (HSQC, COSY, HMBC, TOCSY, NOESY) with parent-structure context.

Who this job is for and why it stalls

Drug-metabolism (MetID) and metabolomics scientists use NMR when LC-MS alone cannot lock down regioselectivity — such as distinguishing which position on an aromatic ring underwent phase I hydroxylation, or confirming the attachment site of a glucuronide or sulfate conjugate.

The bottleneck is that biochemical candidate spaces are full of isobaric lookalikes and endogenous background compounds. Relying on a 1H database match without checking 13C shift residuals across the modified site can burn days chasing an endogenous lookalike or the wrong regioisomer.

How the Spectra workflow runs

  1. Upload enriched metabolite 1H and 13C — Submit the NMR of the isolated or concentrated metabolite in D2O, DMSO-d6, or CD3OD, and include the parent xenobiotic structure or molecular formula when available.
  2. Rank regioisomers and biotransformation candidates — Spectra retrieves candidate structures and compares predicted versus observed chemical shifts across both the parent core and the modified positions.
  3. Evaluate carbon residuals at the site of metabolism — Use 13C RMSD and atom-level residuals to discard isobaric analogues whose carbon shifts conflict with the observed oxidation, dealkylation, or conjugation site.
  4. Inspect unexplained signals — Check unassigned peaks to see whether a conjugate moiety (such as a sugar or amino-acid fragment) or co-isolated matrix component is present.

What to look for in the review

  • Clear 13C separation between competing regioisomers: helps prioritize which synthetic metabolite standard to order or synthesize first.
  • Core matches well, but extra aliphatic/anomeric peaks remain unexplained: points toward a phase II conjugate or co-eluting endogenous component that requires targeted 2D (HSQC/HMBC) confirmation.

Related: unknown compound identification, evidence and confidence, limitations.

Scope & limitations

Where this workflow stops

Spectra does not automate pathway mapping, quantitative metabolomics, or MSI identification levels. Raw biofluids with heavy protein/lipid overlap and 1H-only profiling mixtures are outside scope; the workflow is designed for isolated or enriched material with 13C data.

Chemist sign-off

Recommended validation

Validate top-ranked hypotheses with an authentic standard or reference-spectrum overlay plus orthogonal MS, following the reporting standards required by your study or regulatory team.

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