A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid
2015; Elsevier BV; Volume: 56; Issue: 6 Linguagem: Inglês
10.1194/jlr.d058487
ISSN1539-7262
AutoresRohini Sidhu, Hui Jiang, Nicole Y. Farhat, Nuria Carrillo, Myra Woolery, Elizabeth A. Ottinger, Forbes D. Porter, Jean E. Schaffer, Daniel S. Ory, Xuntian Jiang,
Tópico(s)Receptor Mechanisms and Signaling
Resumo24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker. 24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker. In the CNS, cholesterol originates almost exclusively from in situ synthesis (1.Dietschy J.M. Turley S.D. Cholesterol metabolism in the brain.Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (728) Google Scholar), while circulating cholesterol is normally prevented from entering the CNS by the blood-brain barrier (2.Björkhem I. Meaney S. Brain cholesterol: long secret life behind a barrier.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 806-815Crossref PubMed Scopus (723) Google Scholar). As cholesterol cannot be eliminated in the CNS, and may be toxic to neurons when in excess, it is secreted from the CNS into the circulation predominantly in the form of its polar metabolite, 24(S)-hydroxycholesterol [24(S)-HC] (3.Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2980) Google Scholar). 24(S)-HC is formed almost exclusively in the brain. The enzymatic conversion of CNS cholesterol to 24(S)-HC, which readily crosses the blood-brain barrier, is the major pathway to eliminate cholesterol and maintain cholesterol homeostasis in brain tissue. The cholesterol 24-hydroxylase (CYP46A1) mediating this conversion is mainly located in neurons (4.Lund 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 (524) Google Scholar). The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of increased cholesterol metabolism in the CNS. Plasma 24(S)-HC is decreased in Alzheimer's disease, vascular dementia, multiple sclerosis, Parkinson's disease, and Huntington's disease, reflecting disease burden, the loss of metabolically active neurons, and the degree of structural atrophy (5.Besga A. Cedazo-Minguez A. Kareholt I. Solomon A. Bjorkhem I. Winblad B. Leoni V. Hooshmand B. Spulber G. Gonzalez-Pinto A. et al.Differences in brain cholesterol metabolism and insulin in two subgroups of patients with different CSF biomarkers but similar white matter lesions suggest different pathogenic mechanisms.Neurosci. Lett. 2012; 510: 121-126Crossref PubMed Scopus (15) Google Scholar, 6.Bretillon L. Lutjohann D. Stahle L. Widhe T. Bindl L. Eggertsen G. Diczfalusy U. Bjorkhem I. Plasma levels of 24S-hydroxycholesterol reflect the balance between cerebral production and hepatic metabolism and are inversely related to body surface.J. Lipid Res. 2000; 41: 840-845Abstract Full Text Full Text PDF PubMed Google Scholar, 7.Bretillon L. Siden A. Wahlund L.O. Lutjohann D. Minthon L. Crisby M. Hillert J. Groth C.G. Diczfalusy U. Bjorkhem I. Plasma levels of 24S-hydroxycholesterol in patients with neurological diseases.Neurosci. Lett. 2000; 293: 87-90Crossref PubMed Scopus (128) Google Scholar, 8.Karrenbauer V.D. Leoni V. Lim E.T. Giovannoni G. Ingle G.T. Sastre-Garriga J. Thompson A.J. Rashid W. Davies G. Miller D.H. et al.Plasma cerebrosterol and magnetic resonance imaging measures in multiple sclerosis.Clin. Neurol. Neurosurg. 2006; 108: 456-460Crossref PubMed Scopus (28) Google Scholar, 9.Kölsch H. Heun R. Kerksiek A. Bergmann K.V. Maier W. Lutjohann D. Altered levels of plasma 24S- and 27-hydroxycholesterol in demented patients.Neurosci. Lett. 2004; 368: 303-308Crossref PubMed Scopus (105) Google Scholar, 10.Leoni V. Mariotti C. Nanetti L. Salvatore E. Squitieri F. Bentivoglio A.R. Bandettini di Poggio M. Piacentini S. Monza D. Valenza M. et al.Whole body cholesterol metabolism is impaired in Huntington's disease.Neurosci. Lett. 2011; 494: 245-249Crossref PubMed Scopus (60) Google Scholar, 11.Leoni V. Mariotti C. Tabrizi S.J. Valenza M. Wild E.J. Henley S.M. Hobbs N.Z. Mandelli M.L. Grisoli M. Bjorkhem I. et al.Plasma 24S-hydroxycholesterol and caudate MRI in pre-manifest and early Huntington's disease.Brain. 2008; 131: 2851-2859Crossref PubMed Scopus (99) Google Scholar, 12.Leoni V. Masterman T. Diczfalusy U. De Luca G. Hillert J. Bjorkhem I. Changes in human plasma levels of the brain specific oxysterol 24S-hydroxycholesterol during progression of multiple sclerosis.Neurosci. Lett. 2002; 331: 163-166Crossref PubMed Scopus (97) Google Scholar, 13.Qureischie H. Heun R. Lutjohann D. Popp J. Jessen F. Ledschbor-Frahnert C. Thiele H. Maier W. Hentschel F. Kelemen P. et al.CETP polymorphisms influence cholesterol metabolism but not Alzheimer's disease risk.Brain Res. 2008; 1232: 1-6Crossref PubMed Scopus (20) Google Scholar, 14.Solomon A. Leoni V. Kivipelto M. Besga A. Oksengard A.R. Julin P. Svensson L. Wahlund L.O. Andreasen N. Winblad B. et al.Plasma levels of 24S-hydroxycholesterol reflect brain volumes in patients without objective cognitive impairment but not in those with Alzheimer's disease.Neurosci. Lett. 2009; 462: 89-93Crossref PubMed Scopus (70) Google Scholar, 15.Teunissen C.E. Dijkstra C.D. Polman C.H. Hoogervorst E.L. von Bergmann K. Lutjohann D. Decreased levels of the brain specific 24S-hydroxycholesterol and cholesterol precursors in serum of multiple sclerosis patients.Neurosci. Lett. 2003; 347: 159-162Crossref PubMed Scopus (79) Google Scholar, 16.Teunissen C.E. Lutjohann D. von Bergmann K. Verhey F. Vreeling F. Wauters A. Bosmans E. Bosma H. van Boxtel M.P. Maes M. et al.Combination of serum markers related to several mechanisms in Alzheimer's disease.Neurobiol. Aging. 2003; 24: 893-902Crossref PubMed Scopus (80) Google Scholar, 17.Zuliani G. Donnorso M.P. Bosi C. Passaro A. Dalla Nora E. Zurlo A. Bonetti F. Mozzi A.F. Cortese C. Plasma 24S-hydroxycholesterol levels in elderly subjects with late onset Alzheimer's disease or vascular dementia: a case-control study.BMC Neurol. 2011; 11: 121Crossref PubMed Scopus (43) Google Scholar). Increased cholesterol turnover (i.e., myelin breakdown or neurodegeneration), which occurs at an early stage in these diseases, appears to be associated with a transient increase of 24(S)-HC efflux and higher plasma or CSF 24(S)-HC concentration (18.Hughes T.M. Rosano C. Evans R.W. Kuller L.H. Brain cholesterol metabolism, oxysterols, and dementia.J. Alzheimers Dis. 2013; 33: 891-911Crossref PubMed Scopus (75) Google Scholar, 19.Leoni V. Caccia C. Potential diagnostic applications of side chain oxysterols analysis in plasma and cerebrospinal fluid.Biochem. Pharmacol. 2013; 86: 26-36Crossref PubMed Scopus (31) Google Scholar). Previously, quantification of 24(S)-HC in biological samples was included in oxysterol and steroid analysis using GC or LC coupled with MS. The measurement of total 24(S)-HC was performed after an alkaline hydrolysis of esterified sterols, and the alkaline hydrolysis step was omitted if only free or unesterified 24(S)-HC was measured. GC-MS is widely used for measurement of oxysterols, such as 24(S)-HC, due to its chromatographic resolving capacity, but this method has limitations such as less sample capacity and long GC run (>15 min) (20.Dzeletovic 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 (474) Google Scholar, 21.Kumar B.S. Chung B.C. Lee Y.J. Yi H.J. Lee B.H. Jung B.H. Gas chromatography-mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats.Anal. Biochem. 2011; 408: 242-252Crossref PubMed Scopus (40) Google Scholar, 22.Matysik S. Klunemann H.H. Schmitz G. Gas chromatography-tandem mass spectrometry method for the simultaneous determination of oxysterols, plant sterols, and cholesterol precursors.Clin. Chem. 2012; 58: 1557-1564Crossref PubMed Scopus (51) Google Scholar). LC-MS/MS was demonstrated as a sensitive, specific, and rapid method for the quantification of 24(S)-HC in biological samples. The atmospheric pressure chemical ionization (APCI) (23.Burkard 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 (74) Google Scholar, 24.Mazalli M.R. Sawaya A.C. Eberlin M.N. Bragagnolo N. HPLC method for quantification and characterization of cholesterol and its oxidation products in eggs.Lipids. 2006; 41: 615-622Crossref PubMed Scopus (34) Google Scholar, 25.DeBarber A.E. Lutjohann D. Merkens L. Steiner R.D. Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol.Anal. Biochem. 2008; 381: 151-153Crossref PubMed Scopus (41) Google Scholar) and atmospheric pressure photoionization (26.Ahonen L. Maire F.B. Savolainen M. Kopra J. Vreeken R.J. Hankemeier T. Myohanen T. Kylli P. Kostiainen R. Analysis of oxysterols and vitamin D metabolites in mouse brain and cell line samples by ultra-high-performance liquid chromatography-atmospheric pressure photoionization-mass spectrometry.J. Chromatogr. A. 2014; 1364: 214-222Crossref PubMed Scopus (25) Google Scholar) allow direct, but less sensitive, analysis of 24(S)-HC without derivatization. Although 24(S)-HC can be detected as the [M+NH4]+ ion in ESI (27.McDonald J.G. Smith D.D. Stiles A.R. Russell D.W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma.J. Lipid Res. 2012; 53: 1399-1409Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 28.Bandaru V. Haughey N.J. Quantitative detection of free 24S-hydroxycholesterol, and 27-hydroxycholesterol from human serum.BMC Neurosci. 2014; 15: 2Crossref PubMed Scopus (19) Google Scholar), the sensitivity was greatly enhanced after derivatization with (2-hydrazinyl-2-oxoethyl) trimethylazanium chloride (Girard reagent) (29.Griffiths W.J. Wang Y. Alvelius G. Liu S. Bodin K. Sjovall J. Analysis of oxysterols by electrospray tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 2006; 17: 341-362Crossref PubMed Scopus (95) Google Scholar, 30.Griffiths W.J. Hornshaw M. Woffendin G. Baker S.F. Lockhart A. Heidelberger S. Gustafsson M. Sjovall J. Wang Y. Discovering oxysterols in plasma: a window on the metabolome.J. Proteome Res. 2008; 7: 3602-3612Crossref PubMed Scopus (63) Google Scholar, 31.Karu K. Hornshaw M. Woffendin G. Bodin K. Hamberg M. Alvelius G. Sjovall J. Turton J. Wang Y. Griffiths W.J. Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols.J. Lipid Res. 2007; 48: 976-987Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 32.Meljon A. Theofilopoulos S. Shackleton C.H. Watson G.L. Javitt N.B. Knolker H.J. Saini R. Arenas E. Wang Y. Griffiths W.J. Analysis of bioactive oxysterols in newborn mouse brain by LC/MS.J. Lipid Res. 2012; 53: 2469-2483Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 33.Roberg-Larsen H. Lund K. Vehus T. Solberg N. Vesterdal C. Misaghian D. Olsen P.A. Krauss S. Wilson S.R. Lundanes E. Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.J. Lipid Res. 2014; 55: 1531-1536Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), N,N-dimethylglycine (34.Jiang X. Ory D.S. Han X. Characterization of oxysterols by electrospray ionization tandem mass spectrometry after one-step derivatization with dimethylglycine.Rapid Commun. Mass Spectrom. 2007; 21: 141-152Crossref PubMed Scopus (74) Google Scholar), picolinic acid (35.Honda A. Yamashita K. Hara T. Ikegami T. Miyazaki T. Shirai M. Xu G. Numazawa M. Matsuzaki Y. Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS.J. Lipid Res. 2009; 50: 350-357Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and 4-(dimethylamino)phenyl isocyanate (36.Ayciriex S. Regazzetti A. Gaudin M. Prost E. Dargere D. Massicot F. Auzeil N. Laprevote O. Development of a novel method for quantification of sterols and oxysterols by UPLC-ESI-HRMS: application to a neuroinflammation rat model.Anal. Bioanal. Chem. 2012; 404: 3049-3059Crossref PubMed Scopus (19) Google Scholar). As 24(S)-HC cannot be differentiated by MS from many positional isomers, chromatographic resolution by a long LC run (≥12 min) in most methods is crucial for analysis of 24(S)-HC. While highly abundant 24(S)-HC in plasma and serum (23.Burkard 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 (74) Google Scholar, 25.DeBarber A.E. Lutjohann D. Merkens L. Steiner R.D. Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol.Anal. Biochem. 2008; 381: 151-153Crossref PubMed Scopus (41) Google Scholar, 27.McDonald J.G. Smith D.D. Stiles A.R. Russell D.W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma.J. Lipid Res. 2012; 53: 1399-1409Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 28.Bandaru V. Haughey N.J. Quantitative detection of free 24S-hydroxycholesterol, and 27-hydroxycholesterol from human serum.BMC Neurosci. 2014; 15: 2Crossref PubMed Scopus (19) Google Scholar, 30.Griffiths W.J. Hornshaw M. Woffendin G. Baker S.F. Lockhart A. Heidelberger S. Gustafsson M. Sjovall J. Wang Y. Discovering oxysterols in plasma: a window on the metabolome.J. Proteome Res. 2008; 7: 3602-3612Crossref PubMed Scopus (63) Google Scholar, 35.Honda A. Yamashita K. Hara T. Ikegami T. Miyazaki T. Shirai M. Xu G. Numazawa M. Matsuzaki Y. Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS.J. Lipid Res. 2009; 50: 350-357Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) has been analyzed by LC-MS/MS and GC-MS (20.Dzeletovic 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 (474) Google Scholar, 21.Kumar B.S. Chung B.C. Lee Y.J. Yi H.J. Lee B.H. Jung B.H. Gas chromatography-mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats.Anal. Biochem. 2011; 408: 242-252Crossref PubMed Scopus (40) Google Scholar, 22.Matysik S. Klunemann H.H. Schmitz G. Gas chromatography-tandem mass spectrometry method for the simultaneous determination of oxysterols, plant sterols, and cholesterol precursors.Clin. Chem. 2012; 58: 1557-1564Crossref PubMed Scopus (51) Google Scholar), measurement of low abundant 24(S)-HC in CSF by GC-MS requires large sample volumes (37.Leoni V. Masterman T. Mousavi F.S. Wretlind B. Wahlund L.O. Diczfalusy U. Hillert J. Björkhem I. Diagnostic use of cerebral and extracerebral oxysterols.Clin. Chem. Lab. Med. 2004; 42: 186-191Crossref PubMed Scopus (101) Google Scholar). Here, we report a sensitive and robust LC-MS/MS method with a total run time of 7.5 min for determination of free 24(S)-HC in human plasma and CSF involving a liquid-liquid extraction and derivatization into nicotinate. The lower limits of quantification (LLOQs) were found to be 1 and 0.025 ng/ml for plasma and CSF, respectively. The validated method has been successfully applied to support a National Institutes of Health (NIH)-sponsored clinical trial of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in Niemann-Pick type C1 (NPC1) patients, in which 24(S)-HC was explored as a pharmacodynamic biomarker (38.Ottinger E.A. Kao M.L. Carrillo-Carrasco N. Yanjanin N. Shankar R.K. Janssen M. Brewster M. Scott I. Xu X. Cradock J. et al.Collaborative development of 2-hydroxypropyl-beta-cyclodextrin for the treatment of Niemann-Pick type C1 disease.Curr. Top. Med. Chem. 2014; 14: 330-339Crossref PubMed Scopus (95) Google Scholar). 24(S)-HC was obtained from Avanti Polar Lipids (Alabaster, AL). 25,26,26,26,27,27,27-[2H7]24(R/S)-hydroxycholesterol (D7-24-HC) was obtained from Medical Isotopes, Inc. (Pelham, NH). Nicotinic acid, N,N′-diisopropylcarbodiimide, 4-(dimethylamino)pyridine, formic acid, ammonium acetate, methyl tert-butyl ether, chloroform, and BSA were obtained from Sigma-Aldrich (St. Louis, MO). All HPLC solvents (methanol and acetonitrile) were HPLC grade and were purchased from EMD Chemicals (Gibbstown, NJ). Milli-Q ultrapure water was prepared in-house with a Milli-Q Integral Water Purification System (Billerica, MA). The HP-β-CD was purchased from Roquette (Lestrem, Cedex, France). Pooled control human plasma (K2EDTA), human CSF, six lots of individual human plasma, and six lots of individual human CSF were purchased from BioChemed Services (Winchester, VA). The HP-β-CD was added to CSF to reach a final concentration of 2.5%. All the stock solutions (1 mg/ml) were prepared in methanol. A working solution containing 10 μg/ml of 24(S)-HC was prepared by the dilution of the stock solution with methanol. The internal standard working solutions for plasma (50 ng/ml of D7-24-HC) and CSF (5 ng/ml of D7-24-HC) were prepared in methanol-water (1:1). Because of the endogenous presence of 24(S)-HC in human plasma and CSF, aqueous solutions of 5% BSA and 2.5% HP-β-CD were used to prepare the calibration standards for plasma and CSF, respectively. Calibration curves were prepared by spiking the 24(S)-HC working solution into 5% BSA and 2.5% HP-β-CD solutions, and preparing serial dilutions that yielded eight calibration standards (1, 2, 5, 10, 20, 50, 100, and 200 ng/ml for plasma assay; 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, and 5 ng/ml for CSF assay). Five percent BSA and 2.5% HP-β-CD solutions served as blanks. The same calibration standards in plasma and CSF were also prepared and used to assess responsiveness in different matrixes, which was evaluated by parallelism between standard curves prepared in biological matrix (plasma and CSF) and surrogate matrix (5% BSA and 2.5% HP-β-CD in water). The pooled-plasma and CSF samples were analyzed to establish the mean concentration of endogenous 24(S)-HC by the LC/MS/MS method. The low quality control (LQC), middle quality control (MQC), high quality control (HQC), and dilution quality control (DQC) samples [endogenous level (+0 ng/ml), endogenous level (+75 ng/ml), endogenous level (+150 ng/ml), and endogenous level (+300 ng/ml) for human plasma assay; endogenous level (+0 ng/ml), endogenous level (+2 ng/ml), endogenous level (+4 ng/ml), and endogenous level (+8 ng/ml) for human CSF assay] were prepared by serial dilution after 24(S)-HC working solution was spiked into blank biological matrix. The LLOQ samples for human plasma (1 ng/ml) and CSF (0.025 ng/ml) were prepared in 5% BSA and 2.5% HP-β-CD solutions, respectively. The 24(S)-HC in the DQC samples was higher than the upper limit of quantification (ULOQ) (200 ng/ml for human plasma; 5 ng/ml for human CSF). The human plasma and CSF DQC samples were diluted 1:4 with 5% BSA and 2.5% HP-β-CD solutions, respectively, prior to extraction. For plasma, standards, quality controls (QCs), and blank or study samples (50 μl) were aliquoted into 10 ml glass test tubes. To each tube, internal standard working solution (50 μl) was added except that methanol-water (1:1) (50 μl) was used for a blank. The tubes were vortexed for approximately 15 s. To each tube was added 200 μl of acidic buffer [50 mM ammonium acetate, 1% formic acid (pH 3)] and 1 ml of methyl tert-butyl ether. The samples were vortexed for approximately 10 min and then centrifuged (approximately 2,200 rpm, 4°C, 5 min). The methyl tert-butyl ether phases (supernatants) were transferred to 1.2 ml glass inserts (VWR, West Chester, PA) after aqueous phases in samples were frozen in a dry-ice/ethanol bath. After methyl tert-butyl ether was evaporated with nitrogen at 35°C, to each insert was added 50 μl of derivitization reagent (a solution of 63 mg of N,N′-diisopropylcarbodiimide, 62 mg of nicotinic acid, and 61 mg of 4-(dimethylamino)pyridine in 5 ml of chloroform). The samples were heated at 50°C for 1 h, and the chloroform was removed with nitrogen at 35°C. The samples were reconstituted with 200 μl of methanol. For CSF, standards, QCs, and blank or study samples (200 μl) were aliquoted into 2 ml glass test tubes. To each tube, internal standard working solution (50 μl) was added except that methanol-water (1:1) (50 μl) was used for a blank. The tubes were vortexed for approximately 15 s. To each tube was added 1 ml of methyl tert-butyl ether. The samples were vortexed for approximately 10 min and then centrifuged (approximately 2,200 rpm, 4°C, 5 min). The methyl tert-butyl ether phases (supernatants) were transferred to 1.2 ml glass inserts (VWR) after aqueous phases in samples were frozen in a dry-ice/ethanol bath. After methyl tert-butyl ether was evaporated with nitrogen at 35°C, to each insert was added 50 μl of derivitization reagent (a solution of 63 mg of N,N′-diisopropylcarbodiimide, 62 mg of nicotinic acid, and 61 mg of 4-(dimethylamino)pyridine in 5 ml of chloroform). The samples were heated at 50°C for 1 h, and the chloroform was removed with nitrogen at 35°C. The samples were reconstituted with 200 μl of methanol. LC-MS/MS analysis was conducted on a Shimadzu (Columbia, MD) Prominence HPLC system coupled with an Applied Biosystems/MDS Sciex (Ontario, Canada) 4000QTRAP mass spectrometer using multiple reaction monitoring (MRM). The HPLC system consisted of a Prominence HPLC system with a CBM-20A system controller, 4 LC-20AD pumps, a SIL-20ACHT autosampler, and a DGU-20A5R degasser. The chromatography was performed using a C18 guard column (4 × 3.0 mm, Phenomenex) as the first dimension at ambient temperature and Eclipse XDB-C18 (3 × 100 mm, 3.5 μm; Agilent, Santa Clara, CA) as the second dimension at 50°C. The compartment of the autosampler was set at 4°C. Supplementary Fig. 1 is a schematic of the column and switching valve arrangement for 2D-LC. For the first dimension LC, mobile phase A (0.1% formic acid in water) and mobile phase B [0.1% formic acid in isopropanol-acetonitrile (1:2)] were operated with a gradient elution as follows: 0–0.6 min 60% B, 0.6–0.7 min 60–100% B, 0.7–5.5 min 100% B, 5.5–5.6 min 100–60% B, and 5.6–7.5 min 60% B at a flow rate of 0.6 ml/min. The solvent gradient for second dimension LC using 0.1% formic acid in water (phase C) and 0.1% formic acid in acetonitrile-methanol (1:4) (phase D) at a flow rate of 0.60 ml/min was as follows: 0–0.9 min 95% D, 0.9–6.0 min 95–100% D, 6.0–6.9 min 100% D, 6.9–7.0 min 100–95% D, and 7.0–7.5 min 95% D. Valve 1 was kept at the A position during 0–0.6 min and 1.2–7.5 min, and at the B position during 0.6–1.2 min. Valve 2 was kept at the A position during 0–5.0 min and 7.0–7.5 min, and at the B position during 5.0–6.9 min. The injection volume was 5 and 10 μl for human plasma and CSF samples, respectively. The ESI source temperature was 600°C; the ESI needle was 5,000 V; the declustering potential was 50 V; the entrance potential was 10 V; and the collision cell exit potential was 10 V. The collision and curtain gas were set at medium and 20, respectively. Both desolvation gas and nebulizing gas were set at 45 l/min. For MRM, the collision energies for mass transitions of m/z 307.2–124.0 [quantifier for 24(S)-HC], m/z 307.2–490.4 [qualifier for 24(S)-HC], and m/z 310.7–124.0 (D7-24-HC, internal standard) were 23, 13, and 23 V, respectively. The dwell time was set at 50 ms for each mass transition. Data were acquired and analyzed by Analyst software (version 1.5.1). Calibration curves were constructed by plotting the corresponding peak area ratios of analyte/internal standard versus the corresponding analyte concentrations using weighted (1/x2) least-squares regression analysis. The linearity response of analytes was assessed over their respective calibration range from three batches of analytical runs. The precision and accuracy of the assay were determined for each analyte at LLOQ, LQC, MQC, and HQC concentration levels in human plasma and CSF over the three batch runs. The DQC was used to assess the dilution integration. These QC concentrations included the known fortified levels added to the plasma or CSF plus the endogenous concentration of analyte. For each QC concentration, analysis was performed in six replicates on each day, except for DQCs for which three replicates were prepared. Precision and accuracy are denoted by percent coefficient of variance (CV) and percent relative error (RE), respectively. The accuracy and precision were required to be within ±15% RE of the nominal concentration and ≤15% CV, respectively, for LQC, MQC, HQC, and DQC samples. The accuracy and precision were required to be within ±20% RE of the nominal concentration and ≤20% CV for LLOQ samples in the intra-batch and inter-batch assays (39.US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, and Center for Veterinary Medicine.Guidance for Industry: Bioanalytical Method Validations. Accessed April 7, 2015, at http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf. 2001; Google Scholar). For 24(S)-HC, long-term storage, freeze/thaw stabilities, and stabilities on the bench-top and in the autosampler were determined at the LQC and HQC concentration levels (n = 3). Long-term storage stability of analyte in human plasma and CSF was tested up to 48 and 34 days upon storage at −80°C, respectively. Bench-top stability was evaluated from human plasma and CSF that were kept on the lab bench at room temperature for 4 h before sample extraction. Freeze/thaw stability was tested by freezing the samples overnight, followed by thawing to room temperature the next day. This process was repeated three times. In the autosampler, stability was tested over three days by injecting the first batch of the validation samples. Stock solution stability was established by quantification of samples from dilution of two stock solutions that had been stored at −20°C for 48 days and at room temperature on the bench for 18 h, respectively, to the final solution (200 ng/ml in methanol). A fresh standard curve was established each time. Samples consisting of calibration standards in duplicate, a blank, a blank with internal standard, QCs (LQC, MQC, and HQC), and unknown clinical samples were analyzed. The standard curve covered the expected unknown sample concentration range, and samples that exceeded the highest standard could be diluted and re-assayed. In the dilution sample re-assay, a DQC in triplicate was also included in the analytical run. The results of the QC samples provided the basis for accepting or rejecting the run according to Food and Drug Administration guidelines (39.US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, and Center for Veterinary Medicine.Guidance for Industry: Bioanalytical Method Validations. Accessed April 7, 2015, at http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf. 2001; Google Scholar). This clinical study was approved by the Institutional Review Board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Permission from guardians and assent, when possible, were obtained from
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