A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma
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 sampl...
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doaj-49bcb26f7a8540fd9ec8e790383f60252021-04-28T07:15:01ZengElsevierJournal of Lipid Research0022-22752012-07-0153713991409A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasmaJeffrey G. McDonald0Daniel D. Smith1Ashlee R. Stiles2David W. Russell3To whom correspondence should be addressed.; Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390We 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.http://www.sciencedirect.com/science/article/pii/S002222752034517Xcholesterol/biosynthesischolesterol/metabolismlipidomicsmass spectrometryoxysterolssteroid hormones |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jeffrey G. McDonald Daniel D. Smith Ashlee R. Stiles David W. Russell |
spellingShingle |
Jeffrey G. McDonald Daniel D. Smith Ashlee R. Stiles David W. Russell A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma Journal of Lipid Research cholesterol/biosynthesis cholesterol/metabolism lipidomics mass spectrometry oxysterols steroid hormones |
author_facet |
Jeffrey G. McDonald Daniel D. Smith Ashlee R. Stiles David W. Russell |
author_sort |
Jeffrey G. McDonald |
title |
A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma |
title_short |
A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma |
title_full |
A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma |
title_fullStr |
A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma |
title_full_unstemmed |
A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma |
title_sort |
comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
2012-07-01 |
description |
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. |
topic |
cholesterol/biosynthesis cholesterol/metabolism lipidomics mass spectrometry oxysterols steroid hormones |
url |
http://www.sciencedirect.com/science/article/pii/S002222752034517X |
work_keys_str_mv |
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