title: Unique pentafluorobenzylation and collision-induced dissociation for specific and accurate GC-MS/MS quantification of the catecholamine metabolite 3,4-dihydroxyphenylglycol (DHPG) in human urine.
abstract: In the human body, the catecholamine norepinephrine is mainly metabolized to 3,4-dihydroxyphenylglycol (DHPG) which therefore serves as an important biomarker for norepinephrine's metabolism. Most data on DHPG concentrations in human plasma and urine has been generated by using HPLC-ECD or GC-MS technologies. Here, we describe a stable-isotope dilution GC-MS/MS method for the quantitative determination of DHPG in human urine using trideutero-DHPG (d(3)-DHPG) as internal standard and a two-step derivatization process with pentafluorobenzyl bromide (PFB-Br) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Two pentafluorobenzyl (PFB) trimethylsilyl (TMS) derivatives were obtained and identified, i.e., two isomeric DHPG-PFB-(TMS)(3) derivatives and the later eluting DHPG-tetrafluorobenzyl-(TMS)(2) derivative, i.e., DHPG-TFB-(TMS)(2). To our knowledge the DHPG-TFB-(TMS)(2) derivative and the underlying reaction have not been reported previously. In this reaction both vicinal aromatic hydroxyl groups of DHPG react with PFB-Br to form a heterocyclic seven-membered [1,4]dioxepin compound. The DHPG-TFB-(TMS)(2) derivative was used for quantitative GC-MS/MS analysis in the electron-capturing negative-ion chemical ionization mode by selected-reaction monitoring of m/z 351 from m/z 401 for DHPG and of m/z 352 from m/z 404 for d(3)-DHPG. Validation experiments on human urine samples spiked with DHPG in a narrow (0-33 nM) and a wide range (0-901 nM) revealed high recovery (86-104%) and low imprecision (RSD; 0.01-2.8%). LOD and relative LLOQ (rLLOQ) values of the method for DHPG were determined to be 76 amol and 9.4%, respectively. In urine of 28 patients suffering from chronic inflammatory rheumatic diseases, DHPG was measured at a mean concentration of 238 nM (38.3 μg/g creatinine). The DHPG concentration in the respective control group of 40 healthy subjects was measured to be 328 nM (39.2 μg/g creatinine). Given the unique derivatization reaction and collision-induced dissociation, and the straightforwardness the present method is highly specific, accurate, precise, and should be useful in clinical settings.
authors.affiliation: Institute of Clinical Pharmacology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. zoerner.alexander@mh-hannover.de
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Zoerner
authors.forename: Karsten
authors.initials: K
authors.lastname: Heusser
authors.forename: Frank M
authors.initials: FM
authors.lastname: Gutzki
authors.forename: Anja
authors.initials: A
authors.lastname: Mitschke
authors.forename: Jens
authors.initials: J
authors.lastname: Tank
authors.forename: Dirk O
authors.initials: DO
authors.lastname: Stichtenoth
authors.forename: Jens
authors.initials: J
authors.lastname: Jordan
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002396:Catechols / Q000652:urine*; D005260:Female; D005464:Fluorobenzenes / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D016014:Linear Models; D008297:Male; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives* / Q000652:urine; D008875:Middle Aged; D012213:Rheumatic Fever / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000379:methods; D055815:Young Adult; D005466:Fluorocarbons
chemical_list: D002396:Catechols; D005464:Fluorobenzenes; D008734:Methoxyhydroxyphenylglycol; C025063:pentafluorobenzyl bromide; D005466:Fluorocarbons; C010117:3,4-dihydroxyphenylglycol