Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage.
We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%). The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.
Orhan
Hilmi
H
Department of Pharmacochemistry, Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Vermeulen
Nico P E
NP
Tump
Cornelis
C
Zappey
Herman
H
Meerman
John H N
JH
D002138:Calibration; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003849:Deoxyguanosine / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D018384:Oxidative Stress; D010649:Phenylalanine / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000032:analysis*
D014443:Tyrosine; D010649:Phenylalanine; D003849:Deoxyguanosine