title: A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma.
abstract: 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.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. jeffrey.mcdonald@utsouthwestern.edu
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: McDonald
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Smith
authors.forename: Ashlee R
authors.initials: AR
authors.lastname: Stiles
authors.forename: David W
authors.initials: DW
authors.lastname: Russell
mesh_terms: D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D013058:Mass Spectrometry; D012632:Secosteroids / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*; D013261:Sterols / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D012632:Secosteroids; D013261:Sterols