{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T09:34:50Z","timestamp":1790501690713,"version":"4.1.0"},"reference-count":33,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2018,12,14]],"date-time":"2018-12-14T00:00:00Z","timestamp":1544745600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM111400"],"award-info":[{"award-number":["GM111400"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM069438"],"award-info":[{"award-number":["GM069438"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM103799"],"award-info":[{"award-number":["GM103799"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Metabolites"],"abstract":"<jats:p>Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at \u221280 \u00b0C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.<\/jats:p>","DOI":"10.3390\/metabo8040093","type":"journal-article","created":{"date-parts":[[2018,12,14]],"date-time":"2018-12-14T04:44:42Z","timestamp":1544762682000},"page":"93","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":39,"title":["Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7857-0236","authenticated-orcid":false,"given":"Cora E.","family":"McHugh","sequence":"first","affiliation":[{"name":"NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3962-6014","authenticated-orcid":false,"given":"Thomas L.","family":"Flott","sequence":"additional","affiliation":[{"name":"NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Casey R.","family":"Schooff","sequence":"additional","affiliation":[{"name":"NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zyad","family":"Smiley","sequence":"additional","affiliation":[{"name":"NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael A.","family":"Puskarich","sequence":"additional","affiliation":[{"name":"Department of Emergency Medicine, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel D.","family":"Myers","sequence":"additional","affiliation":[{"name":"Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John G.","family":"Younger","sequence":"additional","affiliation":[{"name":"Akadeum Life Sciences, Ann Arbor, MI 48103, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alan E.","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Emergency Medicine, University of Mississippi, Jackson, MS 39216, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0238-7774","authenticated-orcid":false,"given":"Kathleen A.","family":"Stringer","sequence":"additional","affiliation":[{"name":"NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2018,12,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Psychogios, N., Hau, D.D., Peng, J., Guo, A.C., Mandal, R., Bouatra, S., Sinelnikov, I., Krishnamurthy, R., Eisner, R., and Gautam, B. (2011). The Human Serum Metabolome. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0016957"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.pnmrs.2016.01.005","article-title":"Applications of NMR spectroscopy to systems biochemistry","volume":"92\u201393","author":"Fan","year":"2016","journal-title":"Prog. Nucl. Magn. Reson. Spectrosc."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1165","DOI":"10.1007\/s00216-012-6188-z","article-title":"Quantitative NMR for bioanalysis and metabolomics","volume":"404","author":"Barding","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1097\/SHK.0000000000000406","article-title":"Whole Blood Reveals More Metabolic Detail of the Human Metabolome than Serum as Measured by (1)H-NMR Spectroscopy: Implications for Sepsis Metabolomics","volume":"44","author":"Stringer","year":"2015","journal-title":"Shock (Augusta Ga.)"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1060","DOI":"10.1038\/nprot.2011.335","article-title":"Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry","volume":"6","author":"Dunn","year":"2011","journal-title":"Nat. Protoc."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/j.ab.2009.11.027","article-title":"A sample preparation protocol for 1H nuclear magnetic resonance studies of water-soluble metabolites in blood and urine","volume":"398","author":"Sheedy","year":"2009","journal-title":"Anal. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1006\/abio.2002.5637","article-title":"The Comparison of Plasma Deproteinization Methods for the Detection of Low-Molecular-Weight Metabolites by 1H Nuclear Magnetic Resonance Spectroscopy","volume":"304","author":"Daykin","year":"2002","journal-title":"Anal. Biochem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1021\/acs.analchem.6b04420","article-title":"Recent Advances in NMR-Based Metabolomics","volume":"89","author":"Gowda","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"748","DOI":"10.1111\/j.1432-2277.2003.tb00235.x","article-title":"Organ-specific response to inhibition of mitochondrial metabolism by cyclosporine in the rat","volume":"16","author":"Serkova","year":"2003","journal-title":"Transpl. Int."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1162","DOI":"10.1016\/j.xphs.2016.12.009","article-title":"Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism","volume":"106","author":"Trexel","year":"2016","journal-title":"J. Pharm. Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.plipres.2017.09.003","article-title":"Applications of nuclear magnetic resonance in lipid analyses: An emerging powerful tool for lipidomics studies","volume":"68","author":"Li","year":"2017","journal-title":"Prog. Lipid Res."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Ravanbakhsh, S., Liu, P., Bjorndahl, T.C., Mandal, R., Grant, J.R., Wilson, M., Eisner, R., Sinelnikov, I., Hu, X., and Luchinat, C. (2015). Accurate, fully-automated NMR spectral profiling for metabolomics. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0132873"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5433","DOI":"10.1021\/ac5005103","article-title":"Quantitating Metabolites in Protein Precipitated Serum Using NMR Spectroscopy","volume":"86","author":"Gowda","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.trac.2007.12.001","article-title":"Quantitative metabolomics using NMR","volume":"27","author":"Wishart","year":"2008","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1007\/s10858-011-9480-x","article-title":"Towards automatic metabolomic profiling of high-resolution one-dimensional proton NMR spectra","volume":"49","author":"Mercier","year":"2011","journal-title":"J. Biomol. NMR"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1021\/ac503651e","article-title":"Expanding the Limits of Human Blood Metabolite Quantitation Using NMR Spectroscopy","volume":"87","author":"Gowda","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"872","DOI":"10.1007\/s11306-014-0746-7","article-title":"Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: A review","volume":"11","author":"Emwas","year":"2015","journal-title":"Metabolomics"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2232","DOI":"10.1002\/elps.201700086","article-title":"A review of blood sample handling and pre-processing for metabolomics studies","volume":"38","author":"Hernandes","year":"2017","journal-title":"Electrophoresis"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2606","DOI":"10.1021\/ac303576b","article-title":"Liquid Chromatography\u2013Tandem Mass Spectrometry-Based Plasma Metabonomics Delineate the Effect of Metabolites\u2019 Stability on Reliability of Potential Biomarkers","volume":"85","author":"Yang","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1373\/clinchem.2012.199257","article-title":"Preanalytical aspects and sample quality assessment in metabolomics studies of human blood","volume":"59","author":"Yin","year":"2013","journal-title":"Clin. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1523","DOI":"10.1007\/s00216-012-6039-y","article-title":"Current trends and challenges in sample preparation for global metabolomics using liquid chromatography-mass spectrometry","volume":"403","author":"Vuckovic","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4620","DOI":"10.1021\/acs.analchem.7b00171","article-title":"Whole Blood Metabolomics by 1H NMR Spectroscopy Provides a New Opportunity to Evaluate Coenzymes and Antioxidants","volume":"89","author":"Gowda","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/j.molliq.2016.05.077","article-title":"Solubility of trisodium citrate in water + methanol mixtures at various temperatures","volume":"221","author":"Soleymani","year":"2016","journal-title":"J. Mol. Liq."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.1007\/s11306-013-0520-2","article-title":"NMR-based metabonomics of bovine blood: An investigation into the effects of long term storage on plasma samples","volume":"9","author":"Trabi","year":"2013","journal-title":"Metabolomics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.1039\/C3AN02188B","article-title":"Human plasma stability during handling and storage: Impact on NMR metabolomics","volume":"139","author":"Pinto","year":"2014","journal-title":"Analyst"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.btre.2015.03.004","article-title":"Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container","volume":"6","author":"Roessl","year":"2015","journal-title":"Biotechnol. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1289\/ehp.1205657","article-title":"Performance in Omics Analyses of Blood Samples in Long-Term Storage: Opportunities for the Exploitation of Existing Biobanks in Environmental Health Research","volume":"121","author":"Hebels","year":"2013","journal-title":"Environ. Health Perspect."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1007\/s10858-011-9486-4","article-title":"1H NMR metabolomics identification of markers of hypoxia-induced metabolic shifts in a breast cancer model system","volume":"49","author":"Weljie","year":"2011","journal-title":"J. Biomol. NMR"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.ab.2008.01.037","article-title":"Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy","volume":"377","author":"Tiziani","year":"2008","journal-title":"Anal. Biochem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"70","DOI":"10.11613\/BM.2013.009","article-title":"The effect of storage time and freeze-thaw cycles on the stability of serum samples","volume":"23","author":"Cuhadar","year":"2013","journal-title":"Biochem. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1177\/117693510500100110","article-title":"Impact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projects","volume":"1","author":"Mitchell","year":"2005","journal-title":"Cancer Inform."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Lacy, P., McKay, R.T., Finkel, M., Karnovsky, A., Woehler, S., Lewis, M.J., Chang, D., and Stringer, K.A. (2014). Signal intensities derived from different NMR probes and parameters contribute to variations in quantification of metabolites. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0085732"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1002\/cpbi.11","article-title":"Using MetaboAnalyst 3.0 for Comprehensive Metabolomics Data Analysis","volume":"55","author":"Xia","year":"2016","journal-title":"Curr. Protoc. Bioinform."}],"container-title":["Metabolites"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-1989\/8\/4\/93\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:33:53Z","timestamp":1760196833000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-1989\/8\/4\/93"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,12,14]]},"references-count":33,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2018,12]]}},"alternative-id":["metabo8040093"],"URL":"https:\/\/doi.org\/10.3390\/metabo8040093","relation":{},"ISSN":["2218-1989"],"issn-type":[{"value":"2218-1989","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,12,14]]}}}