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

In the human body, the catecholamine norepinephrine is mainly metabolized to 3,4-dihydroxyphenylglycol (DHPG) which therefore serves as an important biomarker for norepinephrineʹs 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 (d3-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 d3-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.