Monitoring of 8-oxo-7,8-dihydro-2′-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

Abstract

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.

Sensitive Detection of 8-Nitroguanine in DNA by Chemical Derivatization Coupled with Online Solid-Phase Extraction LC-MS/MS

“…The results revealed that an excess of MTNG is required (MTNG:8-nitroG molar ratio 3740:1, Figure 2 ) to completely derivatize 8-nitroG in DNA hydrolysates. This may be attributable to the fact that MTNG reacts not only with 8-nitroG but also with other guanine compounds present in biological samples [ 24 ].…”

“…Fifty-microliter DNA samples were spiked with 50 μL of 5 nM [ 13 C 2 , 15 N]-8-nitroG and subjected to acid hydrolysis in 100 μL of 1 N HCl for 30 min at 80 °C, followed by neutralization with 100 μL of 1 N NaOH. 8-NitroG derivatization and reaction buffer used were described previously [ 24 , 27 , 35 ] and was performed with some modifications. Reaction buffer was prepared by combining 10 mL of 20 mM sodium acetate buffer (pH 4.8), 1 mL of 3 M sodium acetate buffer (pH 5.1) and 1 mL of 1 M Tris-HCl buffer (pH 8.0), aliquoted and stored at −20 °C.…”