To setup and evaluate a high performance liquid chromatography with tandem mass spectrometry assay for urinary 8-hydroxydeoxyguanosine (8-OHdG) analysis and to investigate the biological variation of 8-OHdG excretion in apparently healthy Chinese subjects

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To setup and evaluate a high performance liquid chromatography with tandem mass spectrometry assay for urinary 8-hydroxydeoxyguanosine (8-OHdG) analysis and to investigate the biological variation of 8-OHdG excretion in apparently healthy Chinese subjects

 

Author: Lee, Kam-fai
Title: To setup and evaluate a high performance liquid chromatography with tandem mass spectrometry assay for urinary 8-hydroxydeoxyguanosine (8-OHdG) analysis and to investigate the biological variation of 8-OHdG excretion in apparently healthy Chinese subjects
Degree: M.Sc.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
High performance liquid chromatography.
Carcinogens -- Analysis.
DNA damage.
DNA -- Analysis.
Urine -- Analysis.
Mass spectrometry.
Department: Dept. of Health Technology and Informatics
Pages: xvi, 111 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2305598
URI: http://theses.lib.polyu.edu.hk/handle/200/4898
Abstract: Oxidative damage to cellular biomolecules in particular DNA, has been proposed to play an important role in mutagenesis, carcinogenesis, age-related diseases and aging. Detection of 8-hydroxydeoxyguanosine (8-OHdG), a major by-product of repair of oxidative DNA damage, is important because of its abundance and mutagenic potential. Urinary 8-OHdG concentration may be a potential biomarker or predictor of age-related diseases as well as a measure of oxidative stress induced by diet, environmental and occupational exposure to DNA damaging agents. In this study, the following work was performed: (1) setup and evaluation of a novel high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for urinary 8-OHdG measurement (2) investigation of the urinary concentration of and biological variation in 8-OHdG excretion in apparently healthy Chinese subjects. A simple, rapid, sensitive and reliable HPLC-MS/MS urinary 8-OHdG assay was setup. Direct injection of 25 uL of urine into HPLC-MS/MS system for analysis was performed, with no sample cleanup procedure needed. The total analysis time per sample (from injection to result being obtained) is 15.5 minutes. The method proved to be linear up to at least 452 nmol/L (r2=0.9998). The absolute limit of detection (LOD) and the LOD in urinary matrix were 1.8 nmol/L and 2.3 nmol/L respectively. The within-run and between-run CVs were < 3.0% (n=6 at each of 3 different 8-OHdG concentrations (25.7, 33.0 and 75.1 nmol/L)) and < 5.1% (n=6 at each of 3 different 8-OHdG concentrations (15.6, 24.1 and 66.9 nmol/L)), respectively. The detection reproducibility of urinary 8-OHdG at room temperature over 22 hours was 6.5% (n-5). The mean recovery was 99.3%-102.2% (n=6) across four different 8-OHdG concentrations (7.1, 14.1, 28.2 and 56.5 nmol/L). The range of basal urinary 8-OHdG in 51 apparently healthy Chinese subjects was from undetectable to 42.5 nmol/L (3.3 nmol/mmol creatinine). The median, mean (SD) was 14.5, 15.4 (9.03) nmol/L (1.7, 1.7 (0.60) nmol/mmol creatinine). In age- and body mass index (BMI)-matched apparently healthy Chinese subjects, urinary 8-OHdG (expressed as nmol/mmol creatinine) in males (1.9 (1.60), n=26) was significantly higher than in females (1.5 (0.55), n=25) (p=0.027). No significant correlation of urinary 8-OHdG with age (r2=0.006; p=0.5923) or with BMI (r2=0.002; p=0.7828) was detected. In addition, while within-individual variation was wide, no significant overall changes in 8-OHdG excretion within a single day (p=0.4073) or over five consecutive days (p=0.3815) were seen. The total analytical variation of determining creatinine-normalized urinary 8-OHdG with the current method was 5.6%. The overall biological between-individual and within-individual CVs of 23 apparently healthy Chinese individuals (11 males and 12 females) were 30.8% and 18.3% respectively. The index of individuality was 0.59 and the reference change value (RCV) was 53.5%. The within-individual biological variations were not related to the sex of the individuals (p=0.1317). Stratification according to gender did not significantly decrease the index of individuality or increase the utility of conventional population-based reference. In conclusion, a specific, sensitive, reproducible, simple, automatic and rapid HPLC-MS/MS urinary 8-OHdG assay was developed that met the desirable analytical performance goals, linearity, LOD, recovery and precision. The method is suitable for large scale clinical and epidemiological studies. We have defined preliminary population reference intervals for Hong Kong Chinese using the method, and these will support further studies using this novel biomarker of oxidative stress.

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