Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry.
A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.
Olsen
Raymond
R
National Institute of Occupational Health, P.O. Box 8149 Dep, N-0033 Oslo, Norway. Raymond.Olsen@stami.no
Ovrebø
Steinar
S
Thorud
Syvert
S
Lundanes
Elsa
E
Thomassen
Yngvar
Y
Greibrokk
Tyge
T
Molander
Paal
P
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002417:Cattle; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D018736:DNA Adducts / Q000032:analysis*; D004785:Environmental Pollutants / Q000032:analysis*; D006037:Glyoxal / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D015415:Biomarkers; D018736:DNA Adducts; D004785:Environmental Pollutants; D006037:Glyoxal