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

Journal Article (Journal Article)

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 Cā‚ā‚ˆ 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 Dā‚‚ and Dā‚ƒ. Application of this method to plasma samples revealed that at least 50 samples could be extracted in a routine day.

Full Text

Duke Authors

Cited Authors

  • McDonald, JG; Smith, DD; Stiles, AR; Russell, DW

Published Date

  • July 2012

Published In

Volume / Issue

  • 53 / 7

Start / End Page

  • 1399 - 1409

PubMed ID

  • 22517925

Pubmed Central ID

  • PMC3371252

Electronic International Standard Serial Number (EISSN)

  • 1539-7262

Digital Object Identifier (DOI)

  • 10.1194/jlr.D022285


  • eng

Conference Location

  • United States