Introduction
Cotinine is a major metabolite of nicotine used as a biomarker for active or passive smoking behavior and the half-life of nicotine and cotinine are approximately 0.5-3 and 15-20 hours respectively [1]. Although various chemicals in tobacco smoke such as carbon monoxide and thiocyanate may be used as biomarkers, sensitivity and specificity of these chemicals in terms of smoking exposure, both active or passive, are inadequate because they may be influenced by a number of factors including dietary factors in humans [2].
Nicotine absorbed into the systemic circulation by the lungs is transformed into cotinine via human metabolism by cytochrome P450, and 10-15% of cotinine generated is eliminated by urinary excretion [2, 3]. Though cotinine can be detectable in various matrices of human, i.e., urine, saliva, and blood, the concentration in urine is 4-6 times higher than those measured in blood or saliva [4,5]. For this reason, the analysis of urine sample is preferred as a method for determining the extent of exposure to smoking. Another merit of analyzing urine is that the sample collection procedure is non-invasive compared with other kinds of samples.
Urinary 8-Hydroxy-2’-deoxyguanosine (8-OHdG) is known as a marker of oxidative stress in humans [6]. The reactive oxygen species generated endogenously or exogenously lead to the damage of lipid, protein, and DNA by increasing oxidative stress [7]. 8-OHdG, an indicative marker for oxidative damage of DNA, is generated by damage at C8 of deoxy-guanosine by hydroxyl radical attack [8]. Afterwards, the damaged deoxy guanosine of DNA is repaired by glycosylase and 8-OHdG, and then released into an extracellular fluid, transferred by blood, and excreted through the urine [9]. The oxidative damage of DNA is reportedly associated with aging, carcinogenesis, and degenerative diseases in humans. Therefore, 8-OHdG is being studied as a marker for various diseases including cancer, diabetes, dementia, and degenerative diseases [6,7,10].
The correlation between smoking and reactive oxygen species has continuously been studied and it was found that smoking increases reactive oxygen species. Cases of the studies included an identified correlation of 8-OHdG as a marker for the incidence of DNA damage, and cotinine as a marker for smoking exposure and the correlation was identified by an analysis of bodily fluid in humans [11,12]. However, a majority of the studies applied a different analytical method for each analyte, and there have been few studies for showing a simultaneous analytical method [13,14].
A quantitative analysis has been conducted by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) with solid phase extraction (SPE)[15] and gas chromatography mass spectrometry (GC-MS) [16] or a high-performance liquid chromatography-ultraviolet (HPLC-UV) [17] with liquid-liquid extraction (LLE) to determine the concentration of cotinine in urine. While an enzyme-linked immunosorbent analysis (ELISA) Kit [6,8] had been commonly used for 8-OHdG, various methods such as HPLC [18,19], LC-MS/MS [20] and GC-MS [21] with SPE have been studied recently. The simultaneous analysis of 8-OHdG and cotinine is efficient since oxidative damage indicators are often used to assess health effects of environmental pollutants requires correction of the degree of smoking.
In this study, the simultaneous analytical method for cotinine and 8-OHdG in urine by using LC/MS/MS were developed and validated. A cross-validation was conducted between Green Cross Laboratories and Eulji University.
Materials and Methods
Chemicals and reagents
The standard compounds, Cotinine and 8-OHdG, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The internal standards (IS) used Cotinine-d3 and 8-OHdG-15N5 were supplied by CDN Isotopes (Quebec, Canada) and Cambridge Isotope Laboratories (Andover, MA, USA).The HPLC-grade acetonitrile and water were purchased from Fisher Scientific Korea Ltd. (Ulsan, Korea). The methanol was obtained from TEDIA (Fairfiled, USA). The mobile phase used ammonium formate and formic acid were supplied from Fluka (Buchs, Switzerland). The cartridge for SPE, Oasis® HLB 96-well plate 30 μm (30 mg) was purchased from Waters (Ireland).
Samples
The 200 samples used in the cross-analysis were selected as each of 100 urine samples of adult and children collected from the research project of National Institute of Food and Drug Safety Evaluation (Base Study for Human Body Monitoring of Harmful Substances, Project No. 09182 KFDA607).
Preparation procedure of samples
One hundred microliters of the IS (cotinine-d3 and 8-OHdG -15N5, 5 ng/mL) and 800 μL of 10 mM ammonium formate in 0.1% formic acid were added to 100 μL of each calibration sample and urine sample, respectively. The SPE cartridge was preconditioned with 1 mL methanol and equilibrated with 1 mL water before sample loading. After loading 900 μL of the sample, the cartridge was washed with 1 mL of water and eluted with 1 mL of methanol. The eluted solution was then evaporated. After evaporation, the mixture was reconstituted using 300 μL of 50% acetonitrile and vortexed. The prepared sample was then filtered (0.2 μm) and injected into the LC-MS/MS.
Analytical procedure with LC-MS/MS
The Agilent 1200 System (Agilent, USA) was used as the HPLC equipment with a Kinetex 2.6 μm, HILIC (100 Å, 150 × 2.1 mm) column. API 4000 was used as a tandem mass spectrometer, and the data processing was performed by an Analyst 1.4.2 program (Applied Biosystems, USA). The mobile phase consisted of (A) acetonitrile and (B) 10 mM ammonium formate in 0.1% formic acid under gradient conditions, and the mobile phase was eluted for 0-2.5 min with 5% B. During the next 2.5 min, the proportion of B changed to a linear gradient from 5% to 40%. It was maintained for 1 min for 40% B and returned to the initial condition. The flow rate was 0.25 mL/min and the injection volume was 2 μL.
The analytes were quantified by multiple reaction monitoring in positive mode. The precursor and product ions of the analytes and compound parameters are listed in Table 1. The source and gas parameters were as follows: curtain gas, 35 psi; nebulizer gas, 55 psi; heater gas, 60 psi. The source temperature was 550 °C, the ion spray voltage was 5500 V, and a collision-activated dissociation value of 5 was used to enhance the sensitivity.
Method validation
The method was validated in terms of specificity, linearity, detection limit, accuracy, and precision. Specificity was compared to a chromatogram of the blank sample and the standard sample for the calibration curve, and linearity was determined as the coefficient of determination (r2) of the calibration curve regression equation. The limit of detection was calculated by multiplying 3.3 by the ratio of the standard error and slope of the calibration curve. The accuracy and precision for inter-day and intra-day were evaluated by the recovery rate and the relative standard deviation obtained by analyzing the urine samples (n=6) after addition of standard material to the low, medium and high concentration (7.5, 80, and 160 ng/mL for cotinine and 3, 20, 40 ng/mL for 8-OHdG).
Cross-Laboratory analysis
A cross-laboratory analysis was conducted between the Green Cross Laboratories and Eulji University after a new simultaneous analytical method for urinary cotinine and 8-OHdG was developed. The same sample preparation and analytical methods developed in Green Cross Laboratories were used in Eulji University. In this case, the reproducibility was evaluated by the use of a Pearson correlation coefficient (r), and by using an intraclass correlation coefficient (ICC). The r and ICC were calculated by identified and matched analytical results, which were measured by the two different laboratories mentioned above. In other words, the ICC value means that the portion of intra-group variance in the total variance and is represented by the Equation [22,23]. In this case, it is interpreted as excellent if the ICC value exceeds 0.75, fair or good with 0.4-0.75, poor with less than 0.4.
Results
Method validation
In this study, a simultaneous analytical method for cotinine and 8-OHdG was developed and validated in terms of specificity, linearity, limit of detection, accuracy, and precision.
As can be seen Fig. 1, The chromatogram of the blank and the lowest concentration of the calibration curve was clearly distinguished. Cotinine is known to be highly cross-contaminated and a peak is observed at the blank, but the level is below the detection limit and can be ignored. Linearity was confirmed by using a calibration curve. The range of the calibration curves were 2.5-200 and 1-50 ng/mL of cotinine and 8-OHdG, respectively. Of course, the regression equations for cotinine in triplicate was y = 0.219 x + 0.0206 (r = 0.9995), and those for 8-OHdG in triplicate was y = 0.299 x + 0.00951 (r = 0.9998). The limit of detection were 0.23 and 0.5 ng/mL for cotinine and 8-OHdG, respectively. The analytical method condition and parameters were compared with the other applicable studies (Table 2). Additionally, the volume of the urine sample used to detect cotinine and 8-OHdG with a moderate sensitivity was identified as relatively lower in the method of this study.
Cross-Laboratory analysis
A cross-validation were conducted between the two laboratories to check for robustness, and were evaluated based on inter-lab variability. The results of the cross-validation were shown in Table 3 and Fig. 2. The two institutions prepared the same 200 urine samples and analyzed with LC-MS/MS instrument of each institution. Green Cross Labs. detected 59 cotinine and 195 8-OHdG samples out of 200 samples. Out of 200 samples in Eulji University, cotinine was found in 54 samples and 8-OHdG in 198 samples. Mean of cotinine in each substance was 618 ng/mL (Green Cross Labs.) and 622 ng/mL (Eulji University), and 8-OHdG was 3.99 ng/mL (Green Cross Labs.) and 2.82 ng/mL (Eulji Univ.)
In the case of samples out of the range of the calibration curve, reanalysis was performed by diluting the sample so that the value could be included in the calibration curve. Considering the notation that both r and ICC values exceeded 0.9 for both cotinine and 8-OHdG, there was a significant correlation between the analytical results of the two laboratories, and for that reason it can be concluded that the newly developed method is considered highly reliable and reproducible.
The urinary level of cotinine and 8-OHdG
Urine samples from 100 children and 100 adults were analyzed in two independent laboratories. The results were averaged and stratified by demographic and behavioral characteristics to identify potential differences in urinary biomarker levels.In children, urinary levels of both cotinine and 8-OHdG showed significant associations with age and gender. Specifically, older children exhibited higher concentrations of cotinine (p = 0.01) and 8-OHdG (p = 0.03) compared to younger children.In terms of gender, boys had significantly higher levels of cotinine (p = 0.01) and 8-OHdG (p = 0.01) than girls (Table 4), suggesting possible sex-specific exposure patterns or metabolic differences.
In adults, both biomarkers were strongly influenced by smoking status. Smokers had significantly elevated levels of cotinine (p = 0.001) and 8-OHdG (p < 0.0001) compared to non-smokers. Parental exposure to tobacco smoke was also associated with increased urinary cotinine (p < 0.0001) and 8-OHdG (p = 0.01), indicating the relevance of secondhand smoke exposure. Additionally, alcohol consumption was found to significantly affect urinary cotinine levels, with higher concentrations observed in individuals who consumed alcohol compared to non-drinkers (p = 0.05) (Table 5). This suggests that alcohol consumption may either co-occur with smoking behaviors or influence nicotine metabolism.
Furthermore, when participants were stratified based on the widely accepted urinary cotinine threshold for smokers (100 ng/mL; Kin et al., 2020), those above the cutoff had significantly higher levels of 8-OHdG (5.23 ng/mL) compared to those below the threshold (3.73 ng/mL), reinforcing the link between tobacco exposure and oxidative DNA damage.
Discussion
In this study, an identical analytical method and equipment were used for a cross-analysis between the Green Cross Laboratories and Eulji University. As a result of cross-analysis of the two institutions, the reproducibility and robustness were demonstrated by a significant similarity of cross-analysis results between the two sites.
In other studies, the limit of detection for cotinine was reported as 0.025 μg/L by Hu et al. (2006), and for 8-OHdG as 0.024 ng/mL by the same study, which are lower than the limits reported in this study [24]. It was used for the pretreatment method of analysis and diluted urine sample. Other studies that followed the purification process (LLE, SPE, etc.) of urine samples showed similar or higher levels than the limit of detection of this study (Table 2). The accuracy and precision for intra-day and intra-day as determined by the recovery rate of the standard solution added to the urine samples were showed in Table 6. additionally, the 8-OHdG concentrations were compared in consideration of the level of smoking exposure, which is based on a cotinine cut-off value [28]. The subjects were grouped by the variable of identified cotinine cut-off values (50 ng/mL) and 8-OHdG concentrations. As a result, the level of 8-OHdG concentration was higher in the subjects who were assumed to be exposed to smoking in both adults and children. This tendency was consistent with previous studies and literature reviews [6,8]. However, in most previous studies, the urinary concentration of creatinine was used to adjust a concentration of 8-OHdG. Therefore, the results of this study could not be directly compared to those of other studies. It is expected though that the trend of 8-OHdG and cotinine concentration could be compared with other studies. Moreover, there are other possibilities of exposure to nicotine in daily routine other than smoking.
The concentrations of 8-OHdG in subjects above and below the generally recognized cutoff of 100 ng/mL cotinine concentration in urine, known for smokers, showed statistically significant differences, consistent with previous research indicating that smoking induces oxidative damage [29].
The simultaneous quantification of cotinine and 8-OHdG provides a dual biomarker approach that allows for comprehensive evaluation of both smoking exposure and its associated oxidative DNA damage in a single analysis. This method is particularly useful in epidemiological studies, biomonitoring of environmental exposures, occupational health assessments, and smoking cessation interventions, where both exposure and health effect markers are needed. Unlike single-marker assays, this approach improves analytical efficiency, reduces sample volume requirements, and provides integrated data for risk assessment.
Conclusions
The developed LC-MS/MS method for simultaneous analysis of cotinine and 8-OHdG in urine showed high specificity, sensitivity, and reproducibility across laboratories. Age, gender, smoking, and alcohol consumption were associated with variations in biomarker levels. The study underscores the utility of these biomarkers in assessing smoking exposure and oxidative stress-related health effects and supports their application in large-scale biomonitoring and public health surveillance programs.
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