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 journalArticle

99 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1399-1409
Number of pages11
JournalJournal of Lipid Research
Volume53
Issue number7
DOIs
StatePublished - Jul 2012

Fingerprint

Secosteroids
Plasma (human)
Sterols
Desmosterol
Chemical analysis
Lanosterol
Ergocalciferols
High Pressure Liquid Chromatography
Plasmas
Cholecalciferol
Methylene Chloride
Mass spectrometers
Methanol
Cholesterol
Biosynthetic Pathways
Solid Phase Extraction
Lipids
Oxysterols

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Endocrinology

Cite this

A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. / McDonald, Jeffrey G.; Smith, Daniel D.; Stiles, Ashlee R.; Russell, David W.

In: Journal of Lipid Research, Vol. 53, No. 7, 07.2012, p. 1399-1409.

Research output: Contribution to journalArticle

@article{12b6f5a6c77a465695b75c04ef3b8378,
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.",
keywords = "Cholesterol/biosynthesis, Cholesterol/metabolism, Lipidomics, Mass spectrometry, Oxysterols, Steroid hormones, Vitamin D",
author = "McDonald, {Jeffrey G.} and Smith, {Daniel D.} and Stiles, {Ashlee R.} and Russell, {David W.}",
year = "2012",
month = "7",
doi = "10.1194/jlr.D022285",
language = "English (US)",
volume = "53",
pages = "1399--1409",
journal = "Journal of Lipid Research",
issn = "0022-2275",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "7",

}

TY - JOUR

T1 - A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma

AU - McDonald, Jeffrey G.

AU - Smith, Daniel D.

AU - Stiles, Ashlee R.

AU - Russell, David W.

PY - 2012/7

Y1 - 2012/7

N2 - 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.

AB - 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.

KW - Cholesterol/biosynthesis

KW - Cholesterol/metabolism

KW - Lipidomics

KW - Mass spectrometry

KW - Oxysterols

KW - Steroid hormones

KW - Vitamin D

UR - http://www.scopus.com/inward/record.url?scp=84862339778&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862339778&partnerID=8YFLogxK

U2 - 10.1194/jlr.D022285

DO - 10.1194/jlr.D022285

M3 - Article

VL - 53

SP - 1399

EP - 1409

JO - Journal of Lipid Research

JF - Journal of Lipid Research

SN - 0022-2275

IS - 7

ER -