title: Simultaneous determination of oxysterols, cholesterol and 25-hydroxy-vitamin D3 in human plasma by LC-UV-MS.
abstract: BACKGROUND
  Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.
  
  
  METHODS
  A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.
  
  
  RESULTS
  Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.
  
  
  CONCLUSIONS
  The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.
authors: Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Rohini
authors: R
authors: Narayanaswamy
authors: Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Vignesh
authors: V
authors: Iyer
authors: Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Prachi
authors: P
authors: Khare
authors: Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Mary Lou
authors: ML
authors: Bodziak
authors: Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Darlene
authors: D
authors: Badgett
authors: Department of Neurology, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Robert
authors: R
authors: Zivadinov
authors: Department of Neurology, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Bianca
authors: B
authors: Weinstock-Guttman
authors: Department of Exercise and Nutrition Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Todd C
authors: TC
authors: Rideout
authors: Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Department of Neurology, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Murali
authors: M
authors: Ramanathan
authors: Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors: Richard W
authors: RW
authors: Browne
mesh_terms: D002112:Calcifediol / Q000097:blood*; D002784:Cholesterol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D010100:Oxygen / Q000378:metabolism; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D013261:Sterols / Q000097:blood*
chemical_list: D013261:Sterols; D002784:Cholesterol; D002112:Calcifediol; D010100:Oxygen