title: Dansylation of unactivated alcohols for improved mass spectral sensitivity and application to analysis of cytochrome P450 oxidation products in tissue extracts.
abstract: Chemical derivatization is useful for improving the ionization characteristics of poorly or nonionizable analytes in liquid chromatography-mass spectrometry (LC-MS). Dansyl chloride has been widely used as a derivatizing reagent for fluorescence detection and for facilitating the MS detection of phenols and amines, but not for general alcohols. A new dansylation method for improving the mass spectral sensitivity of unactivated alcohols was developed. The dansylated derivative was formed after incubation of the test compound cholesterol and excess dansyl chloride in CH(2)Cl(2) in the presence of 4-(dimethylamino)-pyridine (DMAP) plus N,N-diisopropylethylamine at 65 °C for 1 h, with an overall yield of 96%. The versatility of dansylation was investigated by utilizing representative lipid compounds (containing different numbers of hydroxy groups) for dansylation. All dansylated derivatives of the selected compounds were detected by LC-MS/MS in the electrospray ionization (ESI) positive ion mode. Validation of the method was established in terms of the sensitivity, stability, and repeatability of dansylation. The method was then applied to characterizing the P450 7A1 oxidation product (dansylated 7α-hydroxycholesterol) in human liver extracts using an LC-MS metabolomics/isotopic labeling approach (Tang, Z.; Guengerich, F. P. Anal. Chem. 2009, 81, 3071-3078). The dansylated derivative of the product was identified, with the signal increased by 10(3)-fold compared with a previous method (derivatization with succinic anhydride and ESI negative ion MS). Quantitation of testosterone in human liver extracts was also done as an example of the application of the dansylation method. Thus, dansylation is a potential method of modifying many alcohols for detection by fluorescence and LC-MS analysis.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, Tennessee 37232-0146, USA.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Tang
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D000438:Alcohols / Q000737:chemistry*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism*; D003619:Dansyl Compounds / Q000737:chemistry*; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D057230:Limit of Detection; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D013739:Testosterone / Q000032:analysis; D014020:Tissue Extracts / Q000378:metabolism*
chemical_list: D000438:Alcohols; D003619:Dansyl Compounds; D006888:Hydroxycholesterols; D014020:Tissue Extracts; D013739:Testosterone; C011724:cholest-5-en-3 beta,7 alpha-diol; D003577:Cytochrome P-450 Enzyme System; D002790:Cholesterol 7-alpha-Hydroxylase; C029038:dansyl chloride
references.citation: J Chromatogr A. 2009 Feb 6;1216(6):956-66
references.pmid: 19118834
references.citation: Rapid Commun Mass Spectrom. 2009 Dec;23(23):3637-46
references.pmid: 19890953
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Dec 1;876(1):76-82
references.pmid: 18993119
references.citation: Anal Chem. 2009 Apr 15;81(8):3071-8
references.pmid: 19301915
references.citation: Rapid Commun Mass Spectrom. 2004;18(14):1621-8
references.pmid: 15282788
references.citation: Eur J Biochem. 1969 Feb;7(4):463-70
references.pmid: 5780484
references.citation: J Biol Chem. 1957 May;226(1):497-509
references.pmid: 13428781
references.citation: Anal Chem. 2009 May 15;81(10):3919-32
references.pmid: 19309105
references.citation: Drug Metab Rev. 2007;39(2-3):581-97
references.pmid: 17786640
references.citation: J Exp Bot. 2005 Jan;56(410):219-43
references.pmid: 15618298
references.citation: Rapid Commun Mass Spectrom. 1998;12(8):419-22
references.pmid: 9586229
references.citation: J Chromatogr A. 2003 May 9;996(1-2):103-13
references.pmid: 12830912
references.citation: Biochemistry. 2005 Mar 8;44(9):3259-71
references.pmid: 15736936
references.citation: Anal Chem. 2007 May 1;79(9):3355-62
references.pmid: 17408243
references.citation: Anal Bioanal Chem. 2004 Feb;378(4):875-82
references.pmid: 14598004
references.citation: J Pharm Biomed Anal. 2008 Jul 15;47(3):560-6
references.pmid: 18313250
references.citation: Mol Interv. 2010 Jun;10(3):153-63
references.pmid: 20539034
references.citation: Rapid Commun Mass Spectrom. 2007;21(13):1973-83
references.pmid: 17526068
references.citation: Rapid Commun Mass Spectrom. 2003;17(5):484-97
references.pmid: 12590398
references.citation: Mass Spectrom Rev. 2007 Jan-Feb;26(1):51-78
references.pmid: 16921475
references.citation: Arch Biochem Biophys. 1998 Feb 15;350(2):324-32
references.pmid: 9473308
references.citation: J Chromatogr A. 2004 Apr 30;1035(1):63-73
references.pmid: 15117075
references.citation: Chembiochem. 2005 Nov;6(11):1941-51
references.pmid: 16206229
references.citation: J Lipid Res. 2008 Sep;49(9):2063-73
references.pmid: 18503032
references.citation: Anal Chem. 2002 Aug 15;74(16):4136-44
references.pmid: 12199585
references.citation: J Pharm Biomed Anal. 2005 Feb 7;37(1):1-18
references.pmid: 15664738