Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high performance liquid chromatography tandem mass spectrometry in human serum

A fast and sensitive high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS) method to identify and quantify oxysterols, phytosterols and non-cholesterol sterols at the pico-molar concentration level in human serum in only one run was developed in this study. This method allows the simultaneous separation and quantitation of individual phytosterols, cholesterol precursors and oxidized derivatives of cholesterol without a derivatization step in a single run, thus providing a more confident quantitation of sterols in serum. After saponification, solid-phase extraction (SPE) used as a clean-up step and HPLC separation, detection by MS was developed using APCI and multiple ion monitoring modes. This method employs reversed-phase C18 SPE cartridges and serum calibrators, as well as isotopically labelled cholesterol as an internal standard added before sample processing. The time consumed for a single sample is reduced from the 4 hours of conventional sterol analysis to 1 hour including the chromatographic run time. The method has been evaluated by analyzing a certified cholesterol sample as well as by comparison to other two methods used as reference, based on GC and enzymatic reaction, respectively. Serum from 14 individuals was successfully analyzed. Detection limits for oxysterols, phytosterols and non-cholesterol sterols, all determined in a single run in small serum volumes, were between 0.47 and 1.69 pM. Intra-day precision was <7% for all sterols in in-house-made lipoprotein-deficient serum. The limits of detection showed high sensitivity with very good intraday- and interday-precision for all sterols. The recoveries of the phytosterols, cholesterol precursors and cholestanol ranged from 77% to 92%. These analytical parameters provide a reliable and reproducible method for the identification and quantitation of human sterols.

[1]  Xi-jun Wang,et al.  Modern analytical techniques in metabolomics analysis. , 2012, The Analyst.

[2]  A. Daneshfar,et al.  Determination of β-sitosterol and cholesterol in oils after reverse micelles with Triton X-100 coupled with ultrasound-assisted back-extraction by a water/chloroform binary system prior to gas chromatography with flame ionization detection. , 2011, Analytica chimica acta.

[3]  G. Corso,et al.  Direct analysis of sterols from dried plasma/blood spots by an atmospheric pressure thermal desorption chemical ionization mass spectrometry (APTDCI-MS) method for a rapid screening of Smith-Lemli-Opitz syndrome. , 2010, The Analyst.

[4]  M. D. Luque de Castro,et al.  Ultrasound-assisted extraction and derivatization of sterols and fatty alcohols from olive leaves and drupes prior to determination by gas chromatography-tandem mass spectrometry. , 2010, Journal of chromatography. A.

[5]  T. Busch,et al.  Artifact generation and monitoring in analysis of cholesterol oxide products. , 2009, Analytical biochemistry.

[6]  E. Ros,et al.  Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes. , 2007, The Journal of clinical endocrinology and metabolism.

[7]  Shihn‐Sheng Wu,et al.  Highly sensitive analysis of cholesterol and sitosterol in foods and human biosamples by liquid chromatography with fluorescence detection. , 2007, Journal of chromatography. A.

[8]  Ying Zhang,et al.  Separation and determination of diversiform phytosterols in food materials using supercritical carbon dioxide extraction and ultraperformance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. , 2007, Analytica chimica acta.

[9]  R. Mensink,et al.  Plasma concentrations of plant sterols: physiology and relationship with coronary heart disease. , 2006, Nutrition reviews.

[10]  M. Eberlin,et al.  HPLC separation and determination of 12 cholesterol oxidation products in fish: comparative study of RI, UV, and APCI-MS detectors. , 2006, Journal of agricultural and food chemistry.

[11]  C. Domeño,et al.  Determination of sterols in biological samples by SPME with on-fiber derivatization and GC/FID , 2005, Analytical and bioanalytical chemistry.

[12]  S. Baumann,et al.  Rapid quantification of free and esterified phytosterols in human serum using APPI-LC-MS/MS Published, JLR Papers in Press, October 16, 2004. DOI 10.1194/jlr.C400004-JLR200 , 2005, Journal of Lipid Research.

[13]  B. Angelin,et al.  Monitoring hepatic cholesterol 7α-hydroxylase activity by assay of the stable bile acid intermediate 7α-hydroxy-4-cholesten-3-one in peripheral blood Published, JLR Papers in Press, January 16, 2003. DOI 10.1194/jlr.D200043-JLR200 , 2003, Journal of Lipid Research.

[14]  F. Violi,et al.  Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status. , 2003, Analytical biochemistry.

[15]  W. Lindner,et al.  Concept of sequential analysis of free and conjugated phytosterols in different plant matrices. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[16]  A. Kuksis.  Plasma non-cholesterol sterols. , 2001, Journal of chromatography. A.

[17]  E. Ros,et al.  Intestinal absorption of triglyceride and cholesterol. Dietary and pharmacological inhibition to reduce cardiovascular risk. , 2000, Atherosclerosis.

[18]  G. Lercker,et al.  Mass spectral fragmentations of cholesterol acetate oxidation products. , 2000, Rapid communications in mass spectrometry : RCM.

[19]  G. Schroepfer,et al.  Sterol synthesis. A timely look at the capabilities of conventional and silver ion high performance liquid chromatography for the separation of C27 sterols related to cholesterol biosynthesis. , 1997, Journal of lipid research.

[20]  U. Diczfalusy,et al.  Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry. , 1995, Analytical biochemistry.

[21]  F. Ursini,et al.  Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass spectrometry. , 1994, Free radical biology & medicine.

[22]  T. Heinemann,et al.  Comparison of intestinal absorption of cholesterol with different plant sterols in man * , 1993, European Journal of Clinical Investigation.

[23]  P. Hylemon,et al.  Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate. , 1989, Analytical biochemistry.

[24]  K. Einarsson,et al.  Correlation between serum levels of some cholesterol precursors and activity of HMG-CoA reductase in human liver. , 1987, Journal of lipid research.

[25]  D. Russell,et al.  Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry. , 2007, Methods in enzymology.

[26]  R. Tilvis,et al.  Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. , 1990, American journal of epidemiology.

[27]  R. Tilvis,et al.  Serum plant sterols and their relation to cholesterol absorption. , 1986, The American journal of clinical nutrition.

[28]  F. McLafferty.  Interpretation of Mass Spectra , 1966 .