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Clinical Chemistry 52: 1381-1388, 2006. First published May 11, 2006; 10.1373/clinchem.2005.063735
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(Clinical Chemistry. 2006;52:1381-1388.)
© 2006 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry with On-Line Solid-Phase Extraction

Chiung-Wen Hu1, Chien-Jen Wang2, Louis W. Chang2 and Mu-Rong Chao3,a

Departments of1 Public Health and3 Occupational Safety and Health, Chung Shan Medical University, Taichung, Taiwan.
2 Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung, Taiwan.

aAddress correspondence to this author at: Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N Road, Taichung, Taiwan 402. Fax 886-4-2324-8194; e-mail mrchao{at}csmu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Quantification of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in urine or blood is used to assess and monitor oxidative stress in patients. We describe the use of on-line solid-phase extraction (SPE) and isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) for automated measurement of urinary 8-oxodGuo.

Methods: Automated purification of urine was accomplished with a switching valve and an Inertsil ODS-3 column. After the addition of 15N5-labeled 8-oxodGuo as an internal standard, urine samples were analyzed within 10 min without sample purification. This method was applied to measure urinary 8-oxodGuo in a group of healthy persons (32 regular smokers and 35 nonsmokers). Urinary cotinine was also assayed by an isotope-dilution LC-MS/MS method.

Results: The lower limit of detection was 5.7 ng/L on column (2.0 fmol). Inter- and intraday imprecision (CV) was <5.0%. Mean recovery of 8-oxodGuo in urine was 99%–102%. Mean (SD) urinary concentrations of 8-oxodGuo in smokers [7.26 (3.14) µg/g creatinine] were significantly higher than those in nonsmokers [4.69 (1.70) µg/g creatinine; P <0.005]. Urinary concentrations of 8-oxodGuo were significantly correlated with concentrations of cotinine in smokers (P <0.05).

Conclusions: This on-line SPE LC-MS/MS method is sufficiently sensitive, precise, and rapid to provide high-throughput direct analysis of urinary 8-oxodGuo without compromising quality and validation criteria. This method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species in living cells have been suggested to be associated with aging, cancer, and some degenerative diseases because they cause oxidative damage to nucleic acids, proteins, and lipids (1). Reactive oxygen species include both oxygen-centered radicals and nonradical compounds. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo; so-called 8-OHdG)1 is the most abundant DNA lesions formed by the addition of the hydroxyl radical to the C-8 position of guanine in DNA. The detection of this lesion is considered important because of its abundance and mutagenic potential, and its concentration could be a good indicator of reactive oxygen species and a potential biomarker of carcinogenesis in vivo (2)(3). Damaged DNA may be repaired by the nucleotide excision repair pathway, and the resulting repair product, 8-oxodGuo, in urine is not affected by diet and cell turnover (4). Moreover, urinary 8-oxodGuo may also originate from the hydrolysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) in the nucleotide pool by the 8-oxodGTPase enzyme, the so-called MTH1 protein (5)(6).

Measurements of urinary 8-oxodGuo have been used to evaluate oxidative stress in patients. Urinary excretion of 8-oxodGuo is increased in patients with lung, bladder, prostate, and breast cancers (7)(8). Similar phenomena have also been found in patients with diabetes, hypertension, acute cardioembolic stroke, and hematologic disorders (9)(10)(11)(12). Higher urinary 8-oxodGuo concentrations have also been detected in neurodegenerative diseases such as Parkinson disease (13). It appears that urinary 8-oxodGuo is useful for indicating cancer risk and other oxidative stress–related diseases (3)(8). Moreover, urinary excretion of 8-oxodGuo can be increased by environmental/occupational exposure to carcinogens (14)(15).

Various analytical techniques have been developed for 8-oxodGuo quantification, including HPLC with electrochemical detection (HPLC-ECD), gas chromatography–mass spectrometry (GC-MS), and ELISA (16)(17)(18). These methods can be difficult to perform in the clinical laboratory and are labor-intensive, require chemical derivatization, and exhibit poor sensitivity or inadequate specificity when used to test urine.

Liquid chromatography–tandem MS (LC-MS/MS) is a powerful technology that can overcome the sensitivity and selectivity issues in analysis of DNA adducts. Accurate quantification of adducted bases at extremely low concentrations has frequently relied on the use of nonradioactive isotope-labeled standards to compensate for the loss of analyte during sample preparation, which has been the most critical step in eliminating the matrix effect for analysis of modified bases by MS (19). Furthermore, on-line sample extraction with a column-switching device is an extremely useful technique for automating the preparation of biological samples for LC-MS methods (20)(21). Its advantages include less ion suppression and relatively short run times as well as higher sensitivity and selectivity, especially for urine samples containing a considerable amount of coeluting interferents.

Numerous studies have suggested that urinary 8-oxodGuo could be a useful biological marker for the assessment of oxidative stress. However, the reference interval for basal urinary 8-oxodGuo excretion is uncertain, with previously reported values from various methods differing widely (3–50 µg/g of creatinine) (18)(22)(23).

We developed an isotope-dilution LC-MS/MS method coupled with on-line solid-phase extraction (SPE) for direct and sensitive analysis of urinary 8-oxodGuo. This method was then applied to investigate the urinary concentrations of 8-oxodGuo in smokers and nonsmokers and their association with cotinine concentrations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals
Solvents and salts were of analytical grade. Reagents were purchased from the following sources: d3-cotinine was from Cambridge Isotope Laboratories, and unlabeled 8-oxodGuo and cotinine were from Sigma. The internal standard, 15N5-8-oxodGuo, was synthesized as described previously (15).

urine samples and participants
Urine samples were obtained from 67 apparently healthy individuals (32 regular smokers and 35 nonsmokers) after 4 days of dietary restrictions. A questionnaire was used to obtain data on age, body mass index (BMI), and smoking status (self-reported daily cigarette consumption). Urine samples were kept at 4 °C during sampling and then stored at –20 °C before analysis. Creatinine was measured by a routine procedure in a local hospital.

on-line spe lc-ms/ms analysis of urinary 8-OXODGUO
Preparation of urine samples.
The urine samples were thawed and thoroughly mixed on a vortex-mixer at room temperature. If a precipitate was present, the urine was diluted 1:1 with deionized water and mixed for 60 s because the precipitates in urine could contain 8-oxodGuo (24). After centrifugation at 5000g for 5 min, 20 µL of urine was diluted 10-fold with 50 mL/L methanol containing 1 mL/L formic acid (FA). To the diluted urine, we added 40 µL of 15N5-8-oxodGuo solution (20 µg/L in 50 mL/L methanol–1 mL/L FA) as internal standard and then mixed the urine on a vortex-mixer for ~5 s. The 8-oxodGuo stock solution was prepared by dissolving 8-oxodGuo in 50 mL/L methanol–1 mL/L FA; it was then serially diluted 1:1 with 50 mL/L methanol–1 mL/L FA to yield aqueous solutions for establishing the calibration curve. Because 8-oxodGuo is usually present in urine, there was no blank matrix available for matrix-matched calibration in this study. However, the use of an isotope-labeled coeluting internal standard should compensate the effects from electrospray ionization suppression by other matrix components.

Automated on-line SPE.
The column-switching system used in this study was as described in detail elsewhere (19). It consisted of a switching valve (2-position microelectric actuator from Valco) and an Inertsil ODS-3 column [50 x 4.6 mm (i.d.); 5 µm bead size]. The switching valve function was controlled by PE-SCIEX control software (Analyst). The column-switching operation, including the LC gradients used during the on-line cleanup and the analytical procedures, is summarized in detail in Table 1 . When the switching valve was at position A, 100 µL of prepared urine sample was loaded on the cartridge by an autosampler (PE series 200; Perkin-Elmer), and a quaternary pump (PE series 200; Perkin-Elmer) delivered the 50 mL/L methanol–1 mL/L FA at a flow rate of 1 mL/min as the loading and washing buffer (solvent Ia). After the column was flushed with the loading buffer for 4 min, the valve switched to the injection position (position B) to inject the sample into the LC system. At 6 min after injection (Table 1 ), the valve was switched back to position A, and the column was eluted with a mobile phase (eluent I) with a linear gradient from 100% solvent Ia to 100% solvent Ib [(500 mL/L methanol–1 mL/L FA for 2 min (min 6 to min 8 after injection; see Table 1 )], followed by 100% solvent Ia for 1 min for equilibration of the column and preparation for the next analysis. The total run time was 10 min.


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  		Table 1. Timetable for the column-switching procedure.

Liquid chromatography.
The HPLC system consisted of 2 series 200 micropumps, a series 200 autosampler (Perkin-Elmer), a Polyamine-II endcapped HPLC column [150 x 4.6 mm (i.d.); 5 µm bead size; YMC], and a guard column [10 x 2 mm (i.d.); YMC]. As shown in Table 1Up , isocratic elution using eluent II was used to separate the analytes. After automatic sample cleanup for 4 min, the sample was automatically eluted from the trap column into the analytical column. The mobile phase was 850 mL/L methanol containing 1 mL/L FA (eluent II) and was delivered at a flow rate of 1 mL/min.

Electrospray ionization MS/MS.
The sample eluting from the HPLC system was introduced into a TurboIonspray source installed on an API 3000 triple-quadrupole mass spectrometer (Applied Biosystems), operated in positive mode with a needle voltage of 5.5 kV, nitrogen as the nebulizing gas, and turbogas temperature set at 500 °C. Data acquisition and quantitative processing were accomplished with AnalystTM software, Ver. 1.1 (Applied Biosystems). The fragmentation pattern of protonated 8-oxodGuo observed in this study (data not shown) was consistent with that reported by Renner et al. (25). For all of the samples, the [M+H]+ ion was selected by the first mass filter. After collisional activation, 2 fragment ions were selected: the most abundant fragment ion, [M+H – 116]+, was used for quantification (quantifier ion), and the second most abundant ion, [M+H – 144]+, was used for qualification (qualifier ion). The dwell times per channel were set at 150 ms for the analyte and 150 ms for the internal standard. Nebulizer and curtain gas flow rates were set at 12 (arbitrary units). The collision-assisted dissociation gas and turbo gas were set at 6 and 8 (arbitrary), respectively. The collision energy was set at 19 eV for the quantifier ion or 45 eV for the qualifier ion with nitrogen as the collision gas. The peak full-width at half-maximum was set to 0.7 Th (Thompson) for both Q1 and Q3.

measurement of urinary cotinine
Urinary cotinine, a major metabolite of nicotine, was measured by an isotope-dilution LC-MS/MS method after a liquid–liquid extraction pretreatment described previously by Chao et al. (20).

statistical methods
We analyzed the data by use of SAS statistical software (Ver. 8.2). Concentrations of urinary 8-oxodGuo and cotinine measured by LC-MS/MS were log-transformed to normalize their distributions before statistical analysis. We used the Student t-test to compare urinary concentrations of 8-oxodGuo and cotinine between nonsmokers and smokers and used Pearson correlation coefficients to study the relationship of urinary 8-oxodGuo with cotinine concentrations or self-reported daily cigarette consumption. We used multiple linear regression models to investigate the relationship of urinary 8-oxodGuo concentrations to cotinine concentrations or daily cigarette consumption after adjusting for other variables (i.e., age and BMI).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
on-line spe lc-ms/ms analysis of 8-OXODGUO in human urine
Chromatography and mass spectra.
A typical on-line SPE LC-MS/MS chromatogram for 8-oxodGuo and 15N5-labeled 8-oxodGuo in the urine of a smoker is shown in Fig. 1 . The MS/MS transitions selected for 8-oxodGuo were m/z 284.1->168.0 for quantification and m/z 284.1->140.0 for qualification; the corresponding transitions for 15N5-8-oxodGuo were m/z 289.1->173.0 for quantification and m/z 289.1->145.0 for qualification.


Figure 1
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  		Figure 1. Chromatograms of urinary 8-oxodGuo in a smoker, as measured by LC-MS/MS coupled with on-line SPE.

(A and B), multiple-reaction monitoring transitions used for quantification; (C and D), transitions used for qualification.

Limits of quantification (LOQ) and detection (LOD).
The LOQ was defined as the lowest 8-oxodGuo concentration that could be reliably and reproducibly measured with values for accuracy, intraday imprecision, and interday imprecision <20%. Using the present method, we determined that the LOQ was 20 ng/L on-column (7.0 fmol in an injection volume of 100 µL), based on direct measurement of diluted calibration solutions. The LOD, defined as the lowest concentration that gave a signal-to-noise ratio of at least 3, was 5.7 ng/L (2.0 fmol in an injection volume of 100 µL).

Linearity, precision, and recovery.
Two linear calibration curves covering the low concentration range (0.019, 0.039, 0.078, 0.156, 0.31, and 0.625 µg/L) and the high concentration range (0.625, 1.25, 2.5, 5, 10, and 20 µg/L) were obtained by serial dilution of aqueous calibrator solutions. Each calibration solution contained 40 µL of 20 µg/L 15N5-8-oxodGuo. Linear regression was calculated with nonweighting and non-zero-forced, and 2 linear equations were obtained: y = 0.1633x – 0.0023 µg/L (r2 = 0.9977) for the low range and y = 0.1471x + 0.02 µg/L (r2 = 0.9997) for the high range. Over the entire concentration range of the calibration curves, the mean observed percentage deviation of back-calculated concentrations was between –4.6% and 9.1% with an imprecision (CV) <10%. We evaluated the precision of the present method by performing replicate determinations of 8-oxodGuo in 3 different urine samples (Table 2 ). The intra- and interday CVs were 2%–3% and 4%–5%, respectively. We determined the recovery of 8-oxodGuo in urine by adding 8-oxodGuo in 5 concentrations (2, 5, 10, 20, and 50 µg/L) to 3 urine samples and measuring 3 replicates of these samples. As shown in Table 2 , the recovery of the present method as calculated from the slope of the regression was 97%–103% (r2 >0.99), and the mean recovery was 99%–102% as estimated from the increase in the measured concentration after addition of 8-oxodGuo divided by the concentration added.


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  		Table 2. Precision and recovery of isotope-dilution LC-MS/MS with on-line SPE for urinary 8-oxodGuo analysis.

Ion suppression.
Ion suppression effects were calculated from the peak areas of the internal standard added to the calibrator solutions and compared with the peak areas of the internal standard that was added to each urinary sample. The relative change in peak area of the internal standard was attributed to matrix effects (26). In this study, the ion suppression effect was <10% for all urine samples.

urinary excretion of 8-OXODGUO and cotinine in smokers and nonsmokers
The characteristics of the participants. and the urinary 8-oxodGuo and cotinine concentrations are summarized in Table 3 . Smokers and nonsmokers were similar in age (mean age, 25.4 and 23.1 years for the smokers and nonsmokers, respectively) and BMI (22.3 and 21.9 kg/m2 for the smokers and nonsmokers, respectively). As for the urinary 8-oxodGuo adjusted for urinary creatinine, smokers had a mean (SD) urinary 8-oxodGuo concentration of 7.26 (3.14) µg/g of creatinine and nonsmokers had a mean concentration of 4.69 (1.70) µg/g of creatinine. Smokers had significantly higher urinary 8-oxodGuo concentrations than did nonsmokers (P <0.005). Moreover, mean urinary cotinine concentrations in smokers were significantly higher than in nonsmokers [1187 (1152) vs 8.5 (5.5) µg/g of creatinine, respectively; P <0.005].


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  		Table 3. Overall characteristics of the study participants.

correlation between urinary 8-OXODGUO and cotinine in smokers
The correlation between urinary 8-oxodGuo and cotinine in smokers is shown in Fig. 2 . Urinary 8-oxodGuo concentrations were associated with urinary cotinine concentrations [Pearson correlation coefficient (r) = 0.43; P <0.05]. We found no significant correlation between urinary 8-oxodGuo and self-reported daily cigarette consumption (Pearson r = 0.02; P = 0.93). Multiple linear regression analysis (Table 4 ) revealed that the correlation between urinary 8-oxodGuo and cotinine was not confounded by age or BMI (P <0.05). There was no correlation between urinary 8-oxodGuo and self-reported daily cigarette consumption after adjustment for age and BMI (P = 0.69).


Figure 2
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  		Figure 2. Correlation between urinary 8-oxodGuo and cotinine in smokers.

Equation for the regression line: y = 0.1573x + 0.3742 (r = 0.43; P <0.05; n = 32).


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  		Table 4. Results of multiple regression analysis for urinary 8-oxodGuo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have developed a rapid, specific, and sensitive isotope-dilution LC-MS/MS method incorporating on-line SPE and an isotopic internal standard that can detect urinary 8-oxodGuo with an LOD of 5.7 ng/L on column (2.0 fmol) and a total analysis time per sample as short as 10 min.

In previous studies, several attempts were made to monitor the urinary excretion of 8-oxodGuo. The most convenient method, a commercial ELISA, generally provided a detection limit of 500 ng/L (~88 fmol in a total assay volume of 50 µL) without prior cleanup. However, to achieve satisfactory specificity and sensitivity for analysis of urine, most developed methods have been combined with tedious manual (off-line) sample cleanup procedures. The HPLC-ECD method described by Germadnik et al. (27) involved 2-step off-line SPE cleanup and had a detection limit of 45 fmol, and the GC-MS method described by Holmberg et al. (17) involved off-line SPE and HPLC purification and enrichment and had a detection limit of 4 fmol. Renner et al. (25) and Ravanat et al. (28) developed LC-MS/MS methods for urinary 8-oxodGuo analysis that included off-line SPE sample cleanup and had LODs of 7 and 20 fmol, respectively. The method established in the present work, which involves on-line sample cleanup/purification coupled to isotope-dilution LC-MS/MS, has a lower LOD (2.0 fmol) than these previously reported methods and provides relatively simple and rapid determination of urinary 8-oxodGuo. Furthermore, this method requires only 20 µL of urine for analysis compared with previous studies, which required ~1–5 mL of urine (16)(17)(27)(28). The requirement of a very small sample volume could allow repeated measurements if a second sample is not available and could reduce the required storage space for samples. Similar progress was also reported previously by Weimann et al. (29), who developed an LC-MS/MS method for determining 8-hydroxylated species of guanine based on direct injection of microliter volumes of urine.

It has been reported that 8-oxodGuo usually exists in serum at concentrations of ~70–200 ng/L (equivalent to ~49–141 fmol in 200 µL of serum) (30)(31) and at ~0.3–4.2 lesions per 106 deoxyguanosines in human DNA (equivalent to ~4.5–63 fmol in 20 µg of DNA) (32). This suggests that our newly developed on-line SPE LC-MS/MS method is also capable of quantifying 8-oxodGuo in either serum or in human DNA and could be useful for monitoring of cancer and other diseases related to oxidative stress. Furthermore, a previous study suggested that artifactual oxidation of deoxyguanosine can occur during sample preparation (33). The on-line SPE LC-MS/MS method in this study eliminates tedious sample purification steps and consequently could decrease the risk of artifactual formation of oxidized bases in DNA samples.

The basal urinary 8-oxodGuo concentration observed in this study was ~4.69 µg/g of creatinine, which is in good agreement with several previous measurements obtained with HPLC-ECD, GC-MS, and LC-MS/MS (3–6 µg/g of creatinine) (22)(27)(28). However, higher basal urinary 8-oxodGuo values with a wide range (10–50 µg/g creatinine) were reportedly obtained with immunoassays such as ELISA (18)(23). It has been suggested that the ELISAs have low specificity and could have overestimated the 8-oxodGuo concentrations in urine. The reasons might include the use in commercial ELISAs of the monoclonal antibody N45.1, which is not sufficiently specific to 8-oxodGuo in urine. This may be particularly relevant for crude urine samples, which contain considerable amounts of cross-reacting substances and other structurally related compounds competing with the N45.1 antibody (i.e., 8-oxo-7,8-dihydroguanosine and 8-oxodGuo containing oligomers) (2). Cigarette smoke contains free radicals in either the gas (nitric oxide) or particle phase (tar radical system). Cigarette smoke also contains a variety of carcinogens and can generate free radicals during metabolic activation (i.e., polycyclic aromatic hydrocarbons) (34). In the present study, smokers had increased mean urinary 8-oxodGuo [7.26 (3.14) µg/g of creatinine] compared with nonsmokers [4.69 (1.70) µg/g of creatinine; Table 3Up ], suggesting that cigarette smoking could induce oxidative stress. Our data are similar to previously reported results from a population-based study showing that smokers excreted ~35% to 50% more 8-oxodGuo in their urine than did nonsmokers (35).

Cotinine is one of the major metabolites of nicotine. Because cotinine has a longer elimination half-life (~20 h compared with 2 h for nicotine), measurement of cotinine in biological fluids has been widely used as a reliable biomarker to estimate active smoking. Previous studies reported that cotinine concentrations in nonsmokers were <30 µg/g of creatinine, whereas concentrations for passive/light smokers (<5 cigarettes/day) and regular smokers were 30–100 and 100–7000 µg/g of creatinine, respectively (36)(37). In the present study, the smokers and nonsmokers had mean urinary cotinine concentrations of 1187 µg/g of creatinine (range, 63–5138 µg/g of creatinine) and 8.5 µg/g of creatinine (range, 1.5–23.1 µg/g of creatinine), respectively, which is consistent with these previously reported ranges. We also found a positive correlation between urinary 8-oxodGuo and cotinine for smokers in this study (r = 0.43; P <0.05; n = 32) that was not confounded by other variables, including age and BMI. Our literature review suggests that this could be the first work to demonstrate a significant dose-dependent relationship between urinary excretion of 8-oxodGuo and nicotine intake. These data may provide a stronger rationale for the higher incidence of some diseases in the cigarette smoking population (i.e., heart disease and lung cancer) (38)(39).

We found no significant correlation between selfreported daily cigarette consumption and urinary 8-oxodGuo concentrations after adjustment for age and BMI (Table 4Up ). One possible explanation for the lack of such a correlation is the inadequacy of self-reported data on smoking status because of recall bias, unwillingness to disclose smoking habits, invalid reported numbers of cigarettes consumed, and the use of various cigarette brands containing oxidants at different concentrations (40)(41).

In conclusion, this study describes a simple, rapid, and reliable LC-MS/MS method for direct determination of urinary 8-oxodGuo. When combined with on-line SPE and isotope dilution, this method could allow for high-throughput analysis of urinary 8-oxodGuo without compromising quality and validation criteria. We showed that this method has a lower LOD (2.0 fmol) than previously reported HPLC-ECD and LC-MS/MS methods and could be suitable for analysis of 8-oxodGuo in human serum and DNA. We found that urinary 8-oxodGuo was significantly correlated with urinary cotinine in smokers, suggesting that cigarette smoke is highly responsible for the increased urinary excretion of 8-oxodGuo. The detection of DNA damage by measurement of adducts in the urine has the advantage of being completely noninvasive compared with tests requiring blood sampling. Despite its higher instrument cost, the on-line SPE LC-MS/MS method could be more cost-effective and more applicable to daily clinical practice; on the basis of the costs for consumables alone, the present method was estimated to cost only US $3.00 per sample compared with approximately US $20.00 per sample for a commercial ELISA. These findings suggest that the present method could enable routine and accurate measurement of urinary 8-oxodGuo for purposes such as large population studies.


   Acknowledgments
 
We acknowledge financial support from the National Science Council, Republic of China (Grant NSC 94-2745-B-040-005-URD). We thank the Division of Environmental Health and Occupational Medicine core facility of the National Health Research Institutes for providing the LC-MS/MS and technical assistance. We also thank Chun-Yen Chien for help in sample preparation.


   Footnotes
 
1 Nonstandard abbreviations: 8-oxodGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine; ECD, electrochemical detection; GC-MS, gas chromatography–mass spectrometry; LC-MS/MS, liquid chromatography–tandem mass spectrometry; SPE, solid-phase extraction; BMI, body mass index; FA, formic acid; LOQ, limit of quantification; and LOD, limit of detection.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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