Feb 20, 2026

NMR Serum Sample Preparation for NMR metabolomics

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5rj6jg1b/v1
  • 1University of Georgia
Deanna Lanier
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5rj6jg1b/v1
Protocol Citation: Deanna Lanier, Mario Uchimiya, Arthur Edison 2026. NMR Serum Sample Preparation for NMR metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rj6jg1b/v1
Manuscript citation:

License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 14, 2024
Last Modified: February 23, 2026
Protocol  Integer ID: 99677
Keywords: nmr serum sample preparation for nmr metabolomic, nmr metabolomics for serum sample, nmr metabolomic, nmr serum sample preparation, serum usingnmr spectroscopy, using methanol extraction, methanol extraction, metabolites in protein, serum sample, analytical chemistry, spectroscopy, serum
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Abstract
This is a modified protocol to conduct NMR metabolomics for serum samples using methanol extraction. This protocol was originally proposed by:

Nagana Gowda, N.G., Raftery,D.Quantitating Metabolites in Protein Precipitated Serum UsingNMR Spectroscopy. Analytical Chemistry 2014,86 (11),5433–5440.


Guidelines
Frozen samples should be thawed in 4 °C cold room or on ice. All sample preparation has to be done in 4 °C cold room or on ice.

Materials
Chemicals and Reagents

  • NaH2PO4, Sodium phosphate monobasic anhydrous
  • HPO4, Sodium phosphate dibasic
  • HCl, Hydrochloric acid
  • NaOH, Sodium hydroxide
  • D2O, Deuteriumoxide, 99.9 atom % D
  • DSS-D6, Sodium 2,2-dimethyl-2-silapentane-5-sulfonate, 98atom % D
  • Methanol
  • Pooled quality control

Equipment
  • alibrated micropipettes (100-µl, 200-µl, and 1000-µl)
  • Pipette tips
  • Labels
  • 250-ml volumetric flask
  • Speed-vac concentrator
  • Vortex mixer
  • Eppendorf centriguge
  • 1.5-ml Eppendorf tubes
  • 5-mm SampleJet NMR tubes (Bruker)
  • Analytical weighing balance
  • pH meter
Before start
Before you start, samples should be randomized and include QA/QC and pooled samples for 2D runs. All tubes should be prelabeled.
Phosphate buffer preparation protocol
In a 250 ml volumetric flask, dissolve 2321.55 mg of anhydrous NaH2PO4 and 802.25 mg of Na2HPO4 in 200 mL of D2O.
Add 833.25 µL of 0.1 DSS-D6 stock solution (1/3 of 1mM DSS when mixed with sample)
Adjust the pH to 7.0 using HCL or NaOH
Adjust the volume to 250 mL with D2O and mix well
Recheck the pH
Store the buffer at 4 °C
NMR Serum Methanol Extraction
Thaw samples on ice or at 4 °C

Add MeOH to samples on ice at a 3:1 methanol-to-sample ratio
  1. 750 µL MeOH 250 µL neat sera for pre-challenge samples
  2. 1200 µL MeOH 400 µL near sera for post-challenge samples



Aliquot 800 µL of sample

Vortex the samples and incubate the samples in -20 °C for 00:20:00
Centrifuge the samples at 16000 rcf for 00:30:00

Transfer the supernatant of each sample to a new tube
Dry the samples in a speed-vac concentrator (time depends on the sample condition)

Store the dry pellets in -80 °C if needed

Thaw the sample on ice or in 4 °C

Add 600 µL of NMR buffer to each sample to resuspend the pellets

Vortex the samples for 00:15:00

Centrifuge the samples for 00:00:10

Transfer 580 µL into 5-mm NMR tube

Protocol references
Nagana Gowda GA, Raftery D. Quantitating metabolites in protein precipitated serum using NMR spectroscopy. Anal Chem. 2014 Jun 3;86(11):5433-40. doi: 10.1021/ac5005103. Epub 2014 May 14. PMID: 24796490; PMCID: PMC4045325.

see also:

Beckonert, O., Keun, H. C., Ebbels, T. M., Bundy, J., Holmes, E., Lindon, J. C., & Nicholson, J. K. (2007). Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nature protocols, 2(11), 2692.