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

2008; Elsevier BV; Volume: 50; Issue: 2 Linguagem: Inglês

10.1194/jlr.d800040-jlr200

ISSN

1539-7262

Autores

Akira Honda, Kouwa Yamashita, Takashi Hara, Tadashi Ikegami, Teruo Miyazaki, Mutsumi Shirai, Guorong Xu, Mitsuteru Numazawa, Yasushi Matsuzaki,

Tópico(s)

Sphingolipid Metabolism and Signaling

Resumo

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. Matsuzaki Y. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS..J. Lipid Res. 2008; 49: 2063-2073Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In this study, we have applied our derivatization method to dihydroxy- and epoxysterols. In each case, singly charged ions were observed as the base peaks in the positive ESI mass spectra and amol levels of these oxysterols were detectable. 4β-Hydroxycholesterol (cholest-5-en-3β,4β-diol), 7α-hydroxycholesterol (cholest-5-en-3β,7α-diol), 22R-hydroxycholesterol (cholest-5-en-3β,22R-diol), 24S-hydroxycholesterol (cholest-5-en-3β,24S-diol), 25-hydroxycholesterol (cholest-5-en-3β,25-diol), and 24S,25-epoxycholesterol (cholest-5-en-24S,25-epoxy-3β-ol) were purchased from Steraloids (Wilton, NH). [25,26,26,26,27,27,27-2H7]4β-hydroxycholesterol, [26,26,26,27,27,27-2H6]24-hydroxycholesterol, [27,27,27-2H3]25-hydroxycholesterol, and [26,26,26,27,27,27-2H6]24,25-epoxycholesterol were obtained from Avanti Polar Lipids (Alabaster, AL). 27-Hydroxycholesterol [(25R)-cholest-5-en-3β,26-diol], [25,26,26,26,27,27,27-2H7]27-hydroxycholesterol, and [25,26,26,26,27,27,27-2H7]7α-hydroxycholesterol were prepared as described previously (24Honda A. Salen G. Matsuzaki Y. Batta A.K. Xu G. Leitersdorf E. Tint G.S. Erickson S.K. Tanaka N. Shefer S. Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27−/− mice and CTX.J. Lipid Res. 2001; 42: 291-300Abstract Full Text Full Text PDF PubMed Google Scholar). Picolinic acid and 2-methyl-6-nitrobenzoic anhydride were purchased from Tokyo Kasei Kogyo (Tokyo, Japan), and 4-dimethylaminopyridine and triethylamine were obtained from Wako Pure Chemical Industries (Osaka, Japan). Additional reagents and solvents were of analytical grade. Blood samples were collected from healthy human volunteers and from a patient with cerebrotendinous xanthomatosis (CTX). After coagulation and centrifugation at 1,500 g for 10 min, serum samples were stored at −20°C until analysis. Informed consent was obtained from all subjects, and the experimental procedures were conducted in accordance with the ethical standards of the Helsinki Declaration. Rat liver microsomes were prepared in our previous study (25Honda A. Mizokami Y. Matsuzaki Y. Ikegami T. Doy M. Miyazaki H. Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS..J. Lipid Res. 2007; 48: 1212-1220Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and had been stored at −70°C until they were used in the present experiments. [2H7]4β-hydroxycholesterol (5 ng), [2H7]7α-hydroxycholesterol (10 ng) [2H6]24-hydroxycholesterol (5 ng), [2H3]25-hydroxycholesterol (1 ng), [2H7]27-hydroxycholesterol (10 ng), and [2H6]24,25-epoxycholesterol (1 ng) as internal standards and 5 μg of butylated hydroxytoluene were added to serum (5 μl) or microsomes (1 mg protein), and saponification was carried out in 0.5 ml of 1 N ethanolic KOH at 37°C for 1 h. After the addition of 0.25 ml of distilled water, sterols were extracted twice with 1 ml of n-hexane, and the extract was evaporated to dryness under a stream of nitrogen. Derivatization to the picolinyl ester was performed according to our previous method (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Matsuzaki Y. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS..J. Lipid Res. 2008; 49: 2063-2073Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) with minor modifications. The reagent mixture for derivatization consisted of 2-methyl-6-nitrobenzoic anhydride (100 mg), 4-dimethylaminopyridine (30 mg), picolinic acid (80 mg), pyridine (1.5 ml), and triethylamine (200 μl). The freshly prepared reagent mixture (170 μl) was added to the sterol extract, and the reaction mixture was incubated at 80°C for 60 min. After the addition of 1 ml of n-hexane, the mixture was vortexed for 30 s and centrifuged at 700 g for 3 min. The clear supernatant was collected and evaporated at 80°C under nitrogen. The residue was redissolved in 50 μl of acetonitrile, and an aliquot (1 μl) was injected into the following LC-MS/MS system. The LC-MS/MS system consisted of a TSQ Quantum Ultra quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an H-ESI probe and a Nanospace SI-2 HPLC system (Shiseido, Tokyo, Japan). Chromatographic separation was performed using a Hypersil GOLD column (150 × 2.1 mm, 3 μm, Thermo Electron) at 40°C, and the following gradient system was used at a flow rate of 300 μl/min: initially, the mobile phase was composed of acetonitrile-methanol-water (40:40:20, v/v/v) containing 0.1% acetic acid; then it was programmed in a linear manner to acetonitrile-methanol-water (45:45:10, v/v/v) containing 0.1% acetic acid over 20 min. The final mobile phase was kept constant for an additional 20 min. The general LC-MS/MS conditions were as follows: spray voltage, 1,000 V; vaporizer temperature, 350°C; sheath gas (nitrogen) pressure, 85 psi; auxiliary gas (nitrogen) flow, 60 arbitrary units; ion transfer capillary temperature, 350°C; collision gas (argon) pressure, 1.5 mTorr; and ion polarity, positive. Selected reaction monitoring (SRM) was conducted using the characteristic precursor-to-product ion transition under optimized collision energy, as listed inTable 1.TABLE 1Positive ESI-MS, MS/MS, SRM, and HPLC data of the picolinoyl ester derivative of each oxysterolSRM DatabThe same HPLC column and flow rate described in Materials and Methods were employed.Oxysterols (Derivatives)MS Data [M+Na]+ (Relative Intensity)MS/MS Dataa[M+Na]+ was used as a precursor ion for each MS/MS analysis. Major product ions were arranged in the order of abundance from left to right. (Collision Energy at Maximum Intensity)Collision EnergyPrecursor to ProductS/NcS/Ns were determined by injecting 100 fg of each derivative.HPLC Datab (RRTdRRTs are expressed relative to the retention time of cholesterol 3β-picolinate.)m/z (%)m/z (V)Vm/z4β-Hydroxycholesterol (cholest-5-en-3β,4β-dipicolinates)635 (100)146 (22)512 (20)22635 → 1462000.777α-Hydroxycholesterol (cholest-5-en-3β,7α-dipicolinates)635 (100)146 (15)–eIntense ion (>5% of base peak) was not observed.15635 → 1462000.6222R-Hydroxycholesterol (cholest-5-en-3β,22R-dipicolinates)635 (100)146 (26)512 (22)22635 → 512400.4524S-Hydroxycholesterol (cholest-5-en-3β,24S-dipicolinates)635 (100)512 (22)146 (31)22635 → 512800.4825-Hydroxycholesterol (cholest-5-en-3β,25-dipicolinates)635 (100)512 (19)146 (28)22635 → 512400.5127-Hydroxycholesterol (cholest-5-en-3β,27-dipicolinates)635 (100)512 (12)146 (33)22635 → 512800.5624S,25-Epoxycholesterol (cholest-5-en-24S,25-epoxy-3β-picolinate)528 (100)146 (20)—e20528 → 146800.41ESI, electrospray ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; RRT, relative retention time; S/N, signal-to-noise ratio; SRM, selected reaction monitoring.a [M+Na]+ was used as a precursor ion for each MS/MS analysis. Major product ions were arranged in the order of abundance from left to right.b The same HPLC column and flow rate described in Materials and Methods were employed.c S/Ns were determined by injecting 100 fg of each derivative.d RRTs are expressed relative to the retention time of cholesterol 3β-picolinate.e Intense ion (>5% of base peak) was not observed. Open table in a new tab ESI, electrospray ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; RRT, relative retention time; S/N, signal-to-noise ratio; SRM, selected reaction monitoring. Data are reported as the mean ± SD. Reproducibility was analyzed by one-way layout (JMP software; SAS Institute Inc., Cary, NC). Recovery was analyzed using a polynomial equation (26Taguchi, G. 1986. Introduction to Quality Engineering-Designing Quality into Products and Process. Asian Productivity Organization, Tokyo, Japan.Google Scholar). Linearity of the calibration curves was analyzed by simple linear regression. Regression analysis was also used to calculate the estimated amount ±95% confidence limit in the recovery study. For all analyses, significance was accepted at the level of P < 0.05. Seven oxysterols were converted into the corresponding picolinyl ester derivatives and positive ESI-MS, MS/MS, SRM, and HPLC data were obtained for each of them (Table 1). All picolinyl ester derivatives exhibited [M+Na]+ ions as the base peaks. The fragmentation pattern of the base peak ion of each derivative was examined under various levels of collision energy, and [M+Na−picolinic acid (C6H5NO2)]+ (m/z = 512) or [picolinic acid (C6H5NO2)+Na]+ (m/z = 146) ions were observed as the most-abundant product ions, so that they were selected as monitoring ions for authentic oxysterols by SRM. The monitoring ions and optimal collision energies for deuterated internal standards were m/z 642 → 146 (22 V) for 3β,4β-dipicolinates of [2H7]4β-hydroxycholesterol, m/z 642 → 146 (15 V) for 3β,7α-dipicolinates of [2H7]7α-hydroxycholesterol, m/z 641 → 518 (22 V) for 3β,24-dipicolinates of [2H6]24-hydroxycholesterol, m/z 638 → 515 (22 V) for 3β,25-dipicolinates of [2H3]25-hydroxycholesterol, m/z 642 → 519 (22 V) for 3β,27-dipicolinates of [2H7]27-hydroxycholesterol, and m/z 534 → 146 (20 V) for 3β-picolinate of [2H6]24,25-epoxycholesterol. To determine the sensitivity of our SRM method, the standard mixture solution of the seven oxysterol derivatives was diluted and injected into the LC-MS/MS system. The limit of detection (signal-to-noise ratio of 3) of each steroid was 2 fg (5 amol) on-column for 4β-hydroxycholesterol and 7α-hydroxycholesterol, 5 fg (12.5 amol) on-column for 24S-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol, and 10 fg (25 amol) on-column for 22R-hydroxycholesterol and 25-hydroxycholesterol. A calibration plot was established for each oxysterol. Different amounts of authentic oxysterol were mixed with deuterated internal standard, derivatized to the picolinyl ester, and quantified as described in the Materials and Methods. The weight ratio of each oxysterol, relative to the corresponding deuterated internal standard, was plotted on the abscissa, and the peak area ratio of the picolinyl ester of the authentic oxysterol to the deuterated variant measured by SRM was plotted on the ordinate. Because deuterium-labeled 22R-hydroxycholesterol was not available, [2H6]24-hydroxycholesterol was used as an internal standard for 22R-hydroxycholesterol. The linearity of the standard curves, as determined by simple linear regression, was excellent, as shown inTable 2.TABLE 2Linearities of calibration plots for each oxysterolOxysterolRange (n)Linear Regression EquationaX is the weight ratio of each oxysterol to the corresponding deuterated internal standard, and Y is the peak area ratio calculated as the peak area of the oxysterol-picolinate(s) divided by that of deuterated oxysterol-picolinate(s) (internal standard). [2H6]24-hydroxycholesterol was used as an internal standard for 22R-hydroxycholesterol.Correlation Coefficient (r)ng4β-Hydroxycholesterol0.05 – 10 (7)Y = 0.436X − 0.0090.9997α-Hydroxycholesterol0.1 – 20 (7)Y = 1.075X − 0.0111.00022R-Hydroxycholesterol0.05 – 5 (6)Y = 0.084X − 0.0000.99324S-Hydroxycholesterol0.05 – 5 (6)Y = 0.615X − 0.0100.99625-Hydroxycholesterol0.01 – 1 (6)Y = 0.935X − 0.0071.00027-Hydroxycholesterol0.1 – 10 (6)Y = 1.400X − 0.0200.99824S,25-Epoxycholesterol0.01 – 2 (7)Y = 0.444X − 0.0040.998a X is the weight ratio of each oxysterol to the corresponding deuterated internal standard, and Y is the peak area ratio calculated as the peak area of the oxysterol-picolinate(s) divided by that of deuterated oxysterol-picolinate(s) (internal standard). [2H6]24-hydroxycholesterol was used as an internal standard for 22R-hydroxycholesterol. Open table in a new tab The separation of various authentic oxysterol picolinates by SRM is shown inFig. 2A. All oxysterol picolinates tested were successfully separated. 7β-Hydroxycholesterol, an autoxidation product of cholesterol, gave a peak just before 7α-hydroxycholesterol (not shown in the figure), and the retention times (relative to cholesterol) of these oxysterols (as picolinates) were 0.61 and 0.62, respectively.Figure 2B–D shows typical SRM chromatograms obtained from 1 mg of protein from rat liver microsomes (Fig. 2B) and 5 μl of sera from a control subject (Fig. 2C) and a CTX patient (Fig. 2D). In rat liver microsomes, a significant amount of 24S,25-epoxycholesterol was detected, whereas only a trace amount of 24S-hydroxycholesterol was observed. In contrast, human serum contained a very low concentration of 24S,25-epoxycholesterol, but a significant amount of 24S-hydroxycholesterol was present. When serum oxysterol profiles were compared between controls and CTX, markedly reduced serum 25- and 27-hydroxycholesterol concentrations were observed. The following studies were performed to determine the precision and accuracy of the present method using rat liver microsomes. Reproducibility was investigated by analyzing four samples in triplicate by LC-MS/MS (Table 3). The results were analyzed by a one-way layout, in which the analytical errors were divided into two sources: sample preparation and SRM measurement. The variances were not considered to be attributable to the sample preparation, because the errors during sample preparation were not significantly larger than those between the measurements (see supplementary Tables I, II). The inter-assay coefficients of variation for the between- and within-sample variations were 1.8% to 12.7% and 2.9% to 11.9%, respectively.TABLE 3Reproducibility of the quantification of each oxysterol in rat liver microsomesRelative SDOxysterolMean ± SD (n = 12)Sample PreparationError (SRM)ng%4β-Hydroxycholesterol5.56 ± 0.283.35.67α-Hydroxycholesterol4.22 ± 0.133.72.922R-Hydroxycholesterol0.107 ± 0.01312.711.924S-Hydroxycholesterol0.104 ± 0.0078.75.825-Hydroxycholesterol0.64 ± 0.021.83.727-Hydroxycholesterol3.16 ± 0.238.16.924S,25-Epoxycholesterol1.11 ± 0.085.18.4Each oxysterol was quantified in 1 mg protein from normal rat liver microsomes. Four samples were prepared and quantified in triplicate by liquid chromatography-tandem mass spectrometry. The results were analyzed by a one-way layout, in which the analytical errors were divided into two sources: sample preparation and SRM measurement. Open table in a new tab Each oxysterol was quantified in 1 mg protein from normal rat liver microsomes. Four samples were prepared and quantified in triplicate by liquid chromatography-tandem mass spectrometry. The results were analyzed by a one-way layout, in which the analytical errors were divided into two sources: sample preparation and SRM measurement. For the recovery experiment, known amounts of oxysterols (a, 2a, 3a; a = 0.05–4.0 ng) were spiked into 1 mg of rat liver microsomal protein (n = 2). After alkaline hydrolysis and derivatization, LC-MS/MS was carried out in triplicate for each sample. The recoveries of the known spiked amounts of the oxysterols ranged from 86.7% to 107.3%, with a mean of 100.6% (Table 4). In addition, the amounts of each endogenous oxysterol found in 1 mg of unspiked microsomal protein were within the 95% confidence limit for the estimated amount of each oxysterol calculated by linear regression analysis; this also constituted an index for the precision and accuracy of the method (see supplementary Table III).TABLE 4Recovery of each oxysterol from rat liver microsomesOxysterolAmount AddedAverage RecoveryaRecovery (%) = (amount found −X0)/amount added × 100. X0 value was obtained fromTABLE 3. (SeeTable 5 in ref.35.) (Mean ± SD) (n = 6)ng%4β-Hydroxycholesterol2.00102.7 ± 8.74.0098.5 ± 9.96.00104.3 ± 11.77α-Hydroxycholesterol4.0089.5 ± 7.18.0086.7 ± 6.912.0090.8 ± 8.822R-Hydroxycholesterol0.05103.0 ± 15.50.10105.2 ± 6.90.1599.8 ± 5.624S-Hydroxycholesterol0.05107.3 ± 14.00.10100.3 ± 8.40.15102.0 ± 9.025-Hydroxycholesterol0.20106.6 ± 12.70.40100.1 ± 6.80.60103.1 ± 5.327-Hydroxycholesterol1.0098.2 ± 15.02.00102.6 ± 4.83.00103.7 ± 2.224S,25-Epoxycholesterol0.4097.5 ± 15.20.80107.2 ± 18.51.20104.2 ± 7.5Known amounts of each oxysterol were spiked into 1 mg protein from normal rat liver microsomes before sample preparation.a Recovery (%) = (amount found −X0)/amount added × 100. X0 value was obtained fromTABLE 3. (SeeTable 5 in ref.35Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Matsuzaki Y. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS..J. Lipid Res. 2008; 49: 2063-2073Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar.) Open table in a new tab Known amounts of each oxysterol were spiked into 1 mg protein from normal rat liver microsomes before sample preparation. Neutral monohydroxysterols are poorly ionized by electrospray. To overcome this disadvantage, we have developed a new method for the enhancement of the ionization efficiency by derivatizing into picolinyl esters (23Honda A. Yamashita K. Miyazaki H. Shirai M. Ikegami T. Xu G. Numazawa M. Hara T. Matsuzaki Y. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS..J. Lipid Res. 2008; 49: 2063-2073Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 27Yamashita K. Kobayashi S. Tsukamoto S. Numazawa M. Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography-electrospray ionization-mass spectrometry.Steroids. 2007; 72: 50-59Crossref PubMed Scopus (59) Google Scholar). Dihydroxy- or epoxysterols are more efficiently ionized by electrospray, and their limit of detection (5–60 fmol on-column) was reported to be more than 10 times lower than that of monohydroxysterols (175–2,000 fmol on-column) (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 paper, we have studied the usefulness of our derivatization method on dihydroxy- and epoxysterols that are key regulatory oxysterols in biol

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