Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

Abstract

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 μl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4β-hydroxycholesterol, 7α-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2–10 fg (5–25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples. We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 μl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4β-hydroxycholesterol, 7α-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2–10 fg (5–25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples. Biological samples contain a large number of oxysterols (1Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google Scholar), and most of them are formed from cholesterol by enzymatic oxidation (2Bodin K. Bretillon L. Aden Y. Bertilsson L. Broome U. Einarsson C. Diczfalusy U. Antiepileptic drugs increase plasma levels of 4β-hydroxycholesterol in humans: evidence for involvement of cytochrome p450 3A4..J. Biol. Chem. 2001; 276: 38685-38689Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 3Pikuleva I.A Cholesterol-metabolizing cytochromes P450..Drug Metab. Dispos. 2006; 34: 513-520Crossref PubMed Scopus (91) Google Scholar, 4Lund E.G Kerr T.A. Sakai J. Li W.P. Russell D.W. cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism.J. Biol. Chem. 1998; 273: 34316-34327Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 5Lund E.G Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc. Natl. Acad. Sci. USA. 1999; 96: 7238-7243Crossref PubMed Scopus (513) Google Scholar, 6Li X. Pandak W.M. Erickson S.K. Ma Y. Yin L. Hylemon P. Ren S. Biosynthesis of the regulatory oxysterol, 5-cholesten-3β,25-diol 3-sulfate, in hepatocytes.J. Lipid Res. 2007; 48: 2587-2596Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) (Fig. 1) or autoxidation (7Smith L. L Cholesterol Autoxidation. Plenum Press, New York1981Crossref Google Scholar). By contrast, the oxysterol 24S,25-epoxycholesterol is not derived from cholesterol but is produced de novo from acetyl-CoA via a shunt in the mevalonate pathway (8Nelson J.A Steckbeck S.R. Spencer T.A. Biosynthesis of 24,25-epoxycholesterol from squalene 2,3;22,23-dioxide..J. Biol. Chem. 1981; 256: 1067-1068Abstract Full Text PDF PubMed Google Scholar). These oxysterols are important molecules for preserving lipid homeostasis in the body. 7α-Hydroxycholesterol is a product of CYP7A1, which is the rate-limiting enzyme in the classic bile acid biosynthetic pathway. 27-Hydroxycholesterol, 24S-hydroxycholesterol, 4β-hydroxycholesterol, 22R-hydroxycholesterol, and 24S,25-epoxycholesterol are effective endogenous ligands of the nuclear receptors liver X receptor α (LXRα) and LXRβ (9Janowski B.A Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXRα..Nature. 1996; 383: 728-731Crossref PubMed Scopus (1429) Google Scholar, 10Janowski B.A Grogan M.J. Jones S.A. Wisely G.B. Kliewer S.A. Corey E.J. Mangelsdorf D.J. Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ..Proc. Natl. Acad. Sci. USA. 1999; 96: 266-271Crossref PubMed Scopus (776) Google Scholar, 11Fu X. Menke J.G. Chen Y. Zhou G. MacNaul K.L. Wright S.D. Sparrow C.P. Lund E.G. 27-Hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells.J. Biol. Chem. 2001; 276: 38378-38387Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). In addition, 27-hydroxycholesterol (12Axelson M. Larsson O. Low density lipoprotein (LDL) cholesterol is converted to 27-hydroxycholesterol in human fibroblasts.J. Biol. Chem. 1995; 270: 15102-15110Abstract Full Text PDF PubMed Scopus (63) Google Scholar), 25-hydroxycholesterol (13Krieger M. Goldstein J.L. Brown M.S. Receptor-mediated uptake of low density lipoprotein reconstituted with 25-hydroxycholesteryl oleate suppresses 3-hydroxy-3-methylglutaryl-coenzyme A reductase and inhibits growth of human fibroblasts.Proc. Natl. Acad. Sci. USA. 1978; 75: 5052-5056Crossref PubMed Scopus (42) Google Scholar), and 24S,25-epoxycholesterol (14Spencer T.A Gayen A.K. Phirwa S. Nelson J.A. Taylor F.R. Kandutsch A.A. Erickson S.K. 24(S),25-Epoxycholesterol. Evidence consistent with a role in the regulation of hepatic cholesterogenesis.J. Biol. Chem. 1985; 260: 13391-13394Abstract Full Text PDF PubMed Google Scholar) are known to downregulate the cholesterol biosynthetic pathway, presumably by blocking the processing of the sterol-regulatory element binding protein. GC-MS has historically been used for the analyses of oxysterols in serum and tissues (1Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google Scholar, 15Breuer O. Björkhem I. Simultaneous quantification of several cholesterol autoxidation and monohydroxylation products by isotope-dilution mass spectrometry.Steroids. 1990; 55: 185-192Crossref PubMed Scopus (62) Google Scholar) because the sensitivity and specificity of conventional GC with flame ionization detector is not sufficient to quantify oxysterols in biological samples. However, GC-MS is still not an ideal method, especially for the analysis of 24S,25-epoxycholesterol, because this epoxycholesterol does not survive the temperature required for GC analysis (16Zhang Z. Li D. Blanchard D.E. Lear S.R. Erickson S.K. Spencer T.A. Key regulatory oxysterols in liver: analysis as Δ4-3-ketone derivatives by HPLC and response to physiological perturbations.J. Lipid Res. 2001; 42: 649-658Abstract Full Text Full Text PDF PubMed Google Scholar). Another approach to quantifying oxysterols in biological samples was HPLC with ultraviolet (UV) detection after derivatization to the Δ4-3-ketones (16Zhang Z. Li D. Blanchard D.E. Lear S.R. Erickson S.K. Spencer T.A. Key regulatory oxysterols in liver: analysis as Δ4-3-ketone derivatives by HPLC and response to physiological perturbations.J. Lipid Res. 2001; 42: 649-658Abstract Full Text Full Text PDF PubMed Google Scholar, 17Ogishima T. Okuda K. An improved method for assay of cholesterol 7α-hydroxylase activity.Anal. Biochem. 1986; 158: 228-232Crossref PubMed Scopus (90) Google Scholar, 18Hylemon P.B Studer E.J. Pandak W.M. Heuman D.M. Vlahcevic Z.R. Chiang Y.L. Simultaneous measurement of cholesterol 7α-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate.Anal. Biochem. 1989; 182: 212-216Crossref PubMed Scopus (68) Google Scholar, 19Teng J.I Smith L.L. High-performance liquid chromatographic analysis of human erythrocyte oxysterols as Δ4-3-ketone derivatives.J. Chromatogr. A. 1995; 691: 247-254Crossref PubMed Scopus (20) Google Scholar). This method made it possible to detect the 24S,25-epoxycholesterol derivative as an intact form, but the lower limit of detection for the Δ4-3-ketones of oxysterols was about 2 ng on-column (16Zhang Z. Li D. Blanchard D.E. Lear S.R. Erickson S.K. Spencer T.A. Key regulatory oxysterols in liver: analysis as Δ4-3-ketone derivatives by HPLC and response to physiological perturbations.J. Lipid Res. 2001; 42: 649-658Abstract Full Text Full Text PDF PubMed Google Scholar), which was not sufficient for quantification of the oxysterols in a small amount of biological sample. Recently, liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) was introduced as a sensitive, specific, and rapid method for the quantification of oxysterols (20Burkard I. Rentsch K.M. von Eckardstein A. Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry.J. Lipid Res. 2004; 45: 776-781Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Saldanha T. Sawaya A.C. Eberlin M.N. Bragagnolo N. HPLC separation and determination of 12 cholesterol oxidation products in fish: comparative study of RI, UV, and APCI-MS detectors.J. Agric. Food Chem. 2006; 54: 4107-4113Crossref PubMed Scopus (91) Google Scholar). In addition, LC-tandem mass spectrometry (LC-MS/MS) using electrospray ionization (ESI) has also been applied to the analysis of oxysterols (22McDonald J.G Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 145-170Crossref PubMed Scopus (120) Google Scholar). In general, ESI is not the best ionization method for neutral steroids because of its poor ionization efficiency. However, our recent study demonstrated that the derivatization of monohydroxysterols into picolinyl esters markedly enhanced the ionization efficiency in the ESI process, and the method was much more sensitive than the assay of native monohydroxysterols by LC-APCI-MS/MS (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Y. sensitive analysis of in human serum by Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). In this applied our derivatization method to and In were as the in the ESI mass and levels of these oxysterols were 25-hydroxycholesterol and 24S,25-epoxycholesterol were from and were from 27-Hydroxycholesterol and were as A. G. Y. A.K. Xu G. E. Erickson S.K. N. S. in hepatic levels of in bile acid between and Lipid Res. 2001; 42: Full Text Full Text PDF PubMed Google Scholar). acid and were from and and were from and were of samples were from human and from a with After and for serum samples were was from and the were in with the of the liver microsomes were in our study A. Y. Y. Ikegami T. M. Miyazaki H. sensitive assay of reductase activity by Lipid Res. 2007; 48: Full Text Full Text PDF PubMed Scopus Google Scholar) and been were used in the (5 (5 (1 and (1 as and of were to serum (5 μl) or microsomes (1 mg protein), and was in of for After the of of sterols were with of and the was to a of to the picolinyl was performed according to our method (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Y. sensitive analysis of in human serum by Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar) with The for derivatization of acid and The μl) was to the and the was for After the of of the was for and for The was and The was in of and an (1 μl) was into the LC-MS/MS The LC-MS/MS of a mass with an and a HPLC separation was performed using a and the was used a of the was of it was in a to acid The was for an The LC-MS/MS were as and was using the as and HPLC of the derivative of HPLC and in and were was used as a for product were in the of from to to were by fg of are to the of cholesterol of was not electrospray mass mass was used as a for product were in the of from to The HPLC and in and were were by fg of are to the of cholesterol of was not in a electrospray mass mass are as the mean was analyzed by one-way layout was analyzed using a polynomial G. to into and Scholar). of the was analyzed by analysis was also used to the amount limit in the recovery was the of oxysterols were converted into the picolinyl derivatives and and HPLC were for of them picolinyl derivatives as the The of the of derivative was levels of and acid = or acid = were as the product that were as for oxysterols by The and for were for of for of for of for of for of and for of the sensitivity of our method, the of the oxysterol derivatives was and into the LC-MS/MS The limit of detection (signal-to-noise ratio of of was 2 fg (5 amol) on-column for 4β-hydroxycholesterol and 7α-hydroxycholesterol, fg amol) on-column for 24S-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol, and fg amol) on-column for and A was for amounts of oxysterol were with derivatized to the picolinyl and as in the and The ratio of oxysterol, to the was the and the ratio of the picolinyl of the oxysterol to the by was the was not was used as an for The of the as by was as of for is the ratio of oxysterol to the and is the ratio calculated as the of the by that of was used as an for = = = = = = 2 = X is the ratio of oxysterol to the and is the ratio calculated as the of the by that of was used as an for in a The separation of oxysterol by is oxysterol were an autoxidation product of a in the and the to of these oxysterols were and from mg of from rat liver microsomes (Fig. and of from a (Fig. and a (Fig. In rat liver a amount of 24S,25-epoxycholesterol was a amount of was In contrast, human serum a low of 24S,25-epoxycholesterol, but a amount of was serum oxysterol were between and markedly serum and 27-hydroxycholesterol were The were performed to the and of the method using rat liver was by samples in by LC-MS/MS 3). The results were analyzed by a one-way in which the were into sample and The variances were not to be to the sample because the sample were not than between the measurements The of for the and were 1.8% to 12.7% and 2.9% to 11.9%, of the quantification of oxysterol in rat liver = oxysterol was in mg from rat liver samples were and in by liquid chromatography-tandem mass The results were analyzed by a one-way in which the were into sample and in a oxysterol was in mg from rat liver samples were and in by liquid chromatography-tandem mass The results were analyzed by a one-way in which the were into sample and the recovery known amounts of oxysterols a = were spiked into mg of rat liver = After alkaline hydrolysis and LC-MS/MS was in for sample. The recoveries of the known spiked amounts of the oxysterols ranged from 86.7% to 107.3%, with a mean of In addition, the amounts of endogenous oxysterol in mg of were the limit for the amount of oxysterol calculated by this also an for the and of the method of oxysterol from rat liver = was in = amounts of oxysterol were spiked into mg from rat liver microsomes sample = was in A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Y. sensitive analysis of in human serum by Lipid Res. Full Text Full Text PDF PubMed Scopus Google in a amounts of oxysterol were spiked into mg from rat liver microsomes sample monohydroxysterols are by this a method for the of the ionization efficiency by into picolinyl esters (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Y. sensitive analysis of in human serum by Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. S. Numazawa M. of derivatives of for liquid ionization-mass spectrometry.Steroids. 2007; PubMed Scopus Google Scholar). or are more by and limit of detection was to be more than lower than that of monohydroxysterols (22McDonald J.G Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 145-170Crossref PubMed Scopus (120) Google Scholar). In this the of our derivatization method and that are key regulatory oxysterols in biological samples. The detection limits of oxysterol and epoxycholesterol were which was about more sensitive than with the ESI method (22McDonald J.G Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 145-170Crossref PubMed Scopus (120) Google Scholar). We also the detection limits of native and by LC-APCI-MS/MS and were about on-column not highly sensitive LC-MS/MS analysis after picolinyl derivatization be used not for monohydroxysterols but also for and A derivatization that are for analysis of been Y. G. S. K. J. of oxysterols by electrospray mass spectrometry.J. 2006; PubMed Scopus Google Scholar) converted oxysterols with a into steroids by using cholesterol and derivatized with the to This method improved the sensitivity by ionization and was applied to the of oxysterols in the K. M. G. K. M. G. J. J. Y. chromatography-mass spectrometry for the of Lipid Res. 2007; 48: Full Text Full Text PDF PubMed Scopus Google Scholar). However, this method has several for and highly sensitive quantification of oxysterols in biological samples. are to into the derivatization and with with an are converted to the and this method the derivative from and which are important in the hepatic bile acid biosynthetic pathway. Recently, and X. X. of oxysterols by electrospray ionization mass spectrometry after derivatization with 2007; PubMed Scopus (72) Google Scholar) method that converted oxysterols into This method to the of the However, was to the esters, and the formed a of are more than of are as for and highly sensitive In our picolinyl Yamashita K. M. Y. S. S. Numazawa M. sensitive determination of and in human serum by liquid ionization mass spectrometry.Steroids. 2007; PubMed Scopus Google Scholar) in a recent study that in the ESI mass In the oxysterols with were also derivatized to picolinyl in the ESI mass which to be a of the picolinyl derivatization of steroids with of the efficiency to the picolinyl and a the detection limits of (5–25 were about lower than of monohydroxysterols (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Y. sensitive analysis of in human serum by Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, our method made it possible to quantify 24S,25-epoxycholesterol in biological samples with high sensitivity and this epoxycholesterol to be of the most important regulatory oxysterols for cholesterol homeostasis B.A Grogan M.J. Jones S.A. Wisely G.B. Kliewer S.A. Corey E.J. Mangelsdorf D.J. Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ..Proc. Natl. Acad. Sci. USA. 1999; 96: 266-271Crossref PubMed Scopus (776) Google Scholar, T.A Gayen A.K. Phirwa S. Nelson J.A. Taylor F.R. Kandutsch A.A. Erickson S.K. 24(S),25-Epoxycholesterol. Evidence consistent with a role in the regulation of hepatic cholesterogenesis.J. Biol. Chem. 1985; 260: 13391-13394Abstract Full Text PDF PubMed Google Scholar), the in biological samples not been because of GC-MS analysis and sensitivity by HPLC with detection (16Zhang Z. Li D. Blanchard D.E. Lear S.R. Erickson S.K. Spencer T.A. Key regulatory oxysterols in liver: analysis as Δ4-3-ketone derivatives by HPLC and response to physiological perturbations.J. Lipid Res. 2001; 42: 649-658Abstract Full Text Full Text PDF PubMed Google Scholar). In this epoxycholesterol in hepatic tissues by GC-MS after derivatization A. G. Y. A.K. Xu G. T. M. S. regulation of X receptor in a with Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the derivative GC several with mass and of 24S,25-epoxycholesterol was this sensitivity that by the method (16Zhang Z. Li D. Blanchard D.E. Lear S.R. Erickson S.K. Spencer T.A. Key regulatory oxysterols in liver: analysis as Δ4-3-ketone derivatives by HPLC and response to physiological perturbations.J. Lipid Res. 2001; 42: 649-658Abstract Full Text Full Text PDF PubMed Google Scholar), it was still not sufficient to quantify this epoxycholesterol in small amounts of biological samples. Another of highly sensitive quantification is that the amount the HPLC be that the was in our In human serum than of oxysterol was the with ng of cholesterol our HPLC this amount of cholesterol was in the and which was from oxysterols and not the separation or of oxysterol HPLC separation was important in the method because oxysterols the and By the the specific of oxysterol was to but than because the and with the The for picolinyl derivatization was the as that in our (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Y. sensitive analysis of in human serum by Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar), but a were the was by using of and the was performed for this but the the of 25-hydroxycholesterol was to picolinyl However, of this was by for After the derivatization were by the of and picolinyl derivatives were in the oxysterol in were by our method and the of 4β-hydroxycholesterol, 7α-hydroxycholesterol, 22R-hydroxycholesterol, 25-hydroxycholesterol, and 24S,25-epoxycholesterol than by However, levels by our method not from by the GC-MS method H. Yamashita H. K. S. and in human serum as an for hepatic bile acid Lipid Res. 1990; Full Text PDF PubMed Google Scholar), and and 24S,25-epoxycholesterol levels to be than the detection limits by the HPLC method K. Smith J. of of derivatives of Lipid Res. 1989; Full Text PDF PubMed Google Scholar). We the that 25-hydroxycholesterol was produced by cholesterol but it is also possible that the was not by the GC-MS This is because the derivative of 25-hydroxycholesterol not an ideal mass in the high mass and was used for the quantification by In general, high is a low mass number is as a for GC-MS analysis of biological samples. We 25-hydroxycholesterol and 4β-hydroxycholesterol by using and and the results been of oxysterols in human with = (2Bodin K. Bretillon L. Aden Y. Bertilsson L. Broome U. Einarsson C. Diczfalusy U. Antiepileptic drugs increase plasma levels of 4β-hydroxycholesterol in humans: evidence for involvement of cytochrome p450 3A4..J. Biol. Chem. 2001; 276: 38685-38689Abstract Full Text Full Text PDF PubMed Scopus (198) Google H. Yamashita H. K. S. and in human serum as an for hepatic bile acid Lipid Res. 1990; Full Text PDF PubMed Google Scholar) GC-MS (1Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google K. Smith J. of of derivatives of Lipid Res. 1989; Full Text PDF PubMed Google (1Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google Scholar) (20Burkard I. Rentsch K.M. von Eckardstein A. Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry.J. Lipid Res. 2004; 45: 776-781Abstract Full Text Full Text PDF PubMed Scopus (72) Google (1Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google (1Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google Scholar) (20Burkard I. Rentsch K.M. von Eckardstein A. Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry.J. Lipid Res. 2004; 45: 776-781Abstract Full Text Full Text PDF PubMed Scopus (72) Google K. Smith J. of of derivatives of Lipid Res. 1989; Full Text PDF PubMed Google liquid chromatography-atmospheric pressure chemical ionization-mass not in a liquid chromatography-atmospheric pressure chemical ionization-mass not A recent study using demonstrated that 25-hydroxycholesterol was also by X. Pandak W.M. Erickson S.K. Ma Y. Yin L. Hylemon P. Ren S. Biosynthesis of the regulatory oxysterol, 5-cholesten-3β,25-diol 3-sulfate, in hepatocytes.J. Lipid Res. 2007; 48: 2587-2596Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). results that not 27-hydroxycholesterol but also 25-hydroxycholesterol were markedly lower in serum from a with with that from a (Fig. which to the that a of the 25-hydroxycholesterol in human serum is derived from In a sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. of and into the picolinyl esters them to be by with sensitivity and This method is for the study of lipid by oxysterols as as the and of in The to the Miyazaki for mass with electrospray ionization liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry liquid chromatography-tandem mass spectrometry liver X receptor α