A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma

Jeffrey G. McDonald, Daniel D. Smith, Ashlee R. Stiles, David W. Russell

Research output: Contribution to journalArticlepeer-review

161 Scopus citations


We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 μl of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ∪ 1 ng/ml of plasma. Extraction efficiency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7α- and 4β-hydroxycholesterol. Intermediates from the cholesterol biosynthetic pathway were also detected, including zymosterol, desmosterol, and lanosterol. This method also allowed the quantification of six secosteroids, including the 25-hydroxylated species of vitamins D2 and D3. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day.

Original languageEnglish (US)
Pages (from-to)1399-1409
Number of pages11
JournalJournal of lipid research
Issue number7
StatePublished - Jul 2012


  • Cholesterol/biosynthesis
  • Cholesterol/metabolism
  • Lipidomics
  • Mass spectrometry
  • Oxysterols
  • Steroid hormones
  • Vitamin D

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology


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