A method for routine quantitation of urinary 8‐hydroxy‐2′‐deoxyguanosine based on solid‐phase extraction and micro‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.

Analysis of 8-hydroxy-2′-deoxyguanosine in human urine using hydrophilic interaction chromatography with tandem mass spectrometry

“…Measurements of 8-OHdG in urine samples are especially well-suited to large-scale human studies and clinical applications because they are noninvasive [10,11]. Urinary 8-OHdG has been analyzed by several methods, such as enzyme-linked immunosorbent assay (ELISA) [12,13], high-performance liquid 4 chromatography with electrochemical detection (HPLC-ECD) [14], gas chromatography with mass spectrometry (GC/MS) [15,16], and liquid chromatography with tandem mass spectrometry (LC/MS/MS) [17][18][19][20][21][22][23][24][25][26]. The ELISA method suffers from the problem of non-selectivity because the antibody may cross-react with other substances present in urine [12,27,28].…”

“…LC-MS/MS, when combined with the isotope dilution technique, is highly selective, sensitive, and accurate, and does not require derivatization [28]. In most of previous LC-MS/MS studies, reversed-phase columns have been used to separate 8-OHdG [17][18][19][20][21][22][23][24][25][26]. As a polar compound, 8-OHdG is hardly retained on a reversed-phase column, even though aqueous mobile phases are used.…”

Analysis of urinary 8-oxo-7,8-dihydro-purine-2’-deoxyribonucleosides by LC-MS/MS and improved ELISA

“…Broadly speaking the approaches to urinary 8-oxodG determination are either chromatographic, such as liquid chromatography with tandem mass spectrometry [6][7][8][9][10][11][12], and high performance liquid chromatography with electrochemical detection [13-Analysis of urinary 8-oxopurines 4 17]; or immunoassay, such as the commercial ELISA kit available from the Japanese Institute for the Control of Aging (JaICA) [18,19]. Immunoassay is clearly the most amenable approach, for most laboratories, not requiring expensive equipment, isotopically-labelled standards or specialist expertise.…”

Neuroendocrine System Response Modulates Oxidative Cellular Damage in Burn Patients

“…Daily urinary excretion of 8OHdG was not markedly changed during the seven days of investigation, although the values were all above the standard value. Standard value of daily total amount of urinary excretion of 8OHdG is 3.0-7.9 μg/day (Sabatini et al 2005), that is 46-125 ng/kg/day for a man weighted 65 kg. day 2 (n = 14) day 3 (n = 11) day 4 (n = 12) day 5 (n = 11) day 6 (n = 12) day 7 (n = 11) epinephrine n.s.…”

“…Within seconds after the stress, enhanced secretion of catecholamines (epinephrine, norepinephrine, and dopamine) from the sympathetic nervous system occurs, and corticotrophin-releasing hormone (CRH) is released into the portal circulation from hypothalamus and enhances secretion of pituitary adrenocorticotrophic hormone (ACTH). In the case of the hemorrhage, the first wave of neuroendocrine response also includes massive secretion of arginin vasopression (AVP) from the pituitary and renin from the kidney (Hench et al 1950;Munck et al 1984;Marzinzig et al 1997;Sabatini et al 2005). From hours to days after the stress, glucocorticoid hormone and sympatho-adreno-medullary system play important roles in coresponding with each other (Ingle 1952;Orchinik et al 1991;Munck and Naray-Fejes-Toth 1992).…”